ANTI-HtrA1 ANTIBODIES AND METHODS OF USE

ABSTRACT

The invention provides anti-HtrA1 antibodies and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/653,201, filed Jul. 18, 2017, which is a continuation of U.S.application Ser. No. 13/651,289, filed Oct. 12, 2012, now U.S. Pat. No.9,738,727, which claims the benefit of U.S. Provisional Application No.61/547,649, filed Oct. 14, 2011, which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing submitted in ASCIIformat via EFS-Web and hereby incorporated by reference in its entirety.Said ASCII copy, created on Sep. 24, 2018, is named50474-115004_Sequence_Listing_09.24.18_ST25.txt and is 47,332 bytes insize.

FIELD OF THE INVENTION

The present invention relates to anti-HtrA1 antibodies and methods ofusing the same.

BACKGROUND

The serine protease HtrA1 (PRSS 11; Clan PA, family S1) belongs to anevolutionarily conserved family of HtrA1 proteins (Clausen, T., et al.,Nat Rev Mol Cell Biol 12:152-62 (2011); Clausen, T., et al., Mol Cell10:443-55 (2002)). In humans, HtrA1, 3, and 4 share the same domainarchitecture: an N-terminal IGFBP-like module and a Kazal-like module, aprotease domain with trypsin-like fold, and a C-terminal PDZ domain. Thephysiological relevance of HtrA1 has been firmly established by theidentification of human loss-of-function mutations causing familialischemic cerebral small-vessel disease (Hara, K., et al., N Engl J Med360:1729-39 (2009)). The molecular mechanism involves deficient TGF-βinhibition by HtrA1 resulting in increased TGF-β signaling (Hara et al.,2009). Dysregulated TGF-β signaling by aberrant HtrA1 expression mayalso contribute to arthritic disease (Oka, C., et al., Development131:1041-53 (2004); Tsuchiya, A., et al., Bone 37:323-36 (2005)),perhaps in conjunction with HtrA1-mediated degradation of variousextracellular matrix components (Chamberland et al., J Biol Chem284:27352-9 (2009); Grau, S., et al., J Biol Chem 281:6124-9 (2006);Hadfield, K. D., et al., J Biol Chem 283:5928-38 (2008); Tocharus, J.,et al., Dev Growth Differ 46:257-74 (2004); Tsuchiya et al., 2005)), orindirectly via up-regulation of matrix metalloproteases (Grau et al.,2006). In addition, human genetic studies identified a strongcorrelation between progression of age-related macular degeneration anda SNP in the HtrA1 promoter region, which results in increased HtrA1transcript levels (Dewan, A., et al., Science 314:989-92 (2006); Yang,Z., et al., Science 314:992-3 (2006)). Therefore, inhibition of HtrA1enzymatic function is an attractive therapeutic approach, e.g. inage-related macular degeneration and in arthritic disease.

SUMMARY

The invention provides anti-HtrA1 antibodies and methods of using thesame for diagnostic and therapeutic purposes.

In one aspect, the invention provides an isolated antibody that binds toHtrA1 competitively with antibody comprising a VH sequence of SEQ IDNO:8 and a VL sequence of SEQ ID NO:7. Competitive binding may bedetermined, for example, using an ELISA assay.

In one aspect, the invention provides an isolated antibody that bind toHtrA1 having one or more of the following properties: (i) an IC₅₀ ofless than 50 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 3 nM, 2.5 nM,2 nM, 1 nM, or less for one or more HtrA1 substrates; (ii) binds toHtrA1 with a ratio of 1 variable domain to one subunit of an HtrA1trimer (e.g., a Fab binds to an HtrA1 trimer with a ratio of 3 Fab to 1HtrA1 trimer, and an IgG binds to an HtrA1 trimer with a ratio of 3 IgGto 2 HtrA1 trimers), (iii) for antibodies comprising two variabledomains, binds to HtrA1 in a manner that results in the forming a “cage”similar to that shown in FIG. 9, (iv) does not prevent trimer formationof HtrA1, (v) binds to one or more residues in Loop C of the HtrA1protein, (vi) binds to the protease domain of HtrA1, (vii) binds to anepitope comprising one or both of amino acids N224 or K248 of SEQ IDNO:13, or amino acids equivalent thereto in a different HtrA1 sequence(e.g., amino acids N224 and K248 of SEQ ID NO:14, see FIGS. 10A and B);(viii) binds to an epitope comprising one or more of the followingresidues of N224, K248, V201, T223, K243, K225, E247 and H220 of SEQ IDNO:13, or amino acids equivalent thereto in a different HtrA1 sequence;(ix) cross-reacts with murine HtrA1; (x) does not cross-react withHtrA2, HtrA3 and/or HtrA4; (xi) binds to HtrA1 competitively with anantibody comprising a VH sequence of SEQ ID NO:8 and a VL sequence ofSEQ ID NO:7, (xii) binds to HtrA1 with a dissociation constant of ≤500nM, or (xiii) inhibits complex formation between HtrA1 andα1-antitrypsin (A1AT).

In another aspect, the invention provides an isolated antibody thatbinds to HtrA1, wherein the antibody (i) binds to an epitope thatincludes N224, K248, or both of HtrA1, and (ii) inhibits HtrA1 with anIC₅₀ of ≤30 nM. In certain embodiments, the epitope further includes oneor more of the following residues of HtrA1: V201, T223, K243, K225, E247and H220. In certain embodiments, the antibody may further comprise oneor more of the following properties: (i) binds to HtrA1 with a ratio of1 variable domain to one subunit of an HtrA1 trimer, or (ii) does notprevent trimer formation of HtrA1. In certain embodiments, the IC₅₀ isdetermined using a serine protease assay with a substrate having SEQ IDNO:12, e.g., such as the FRET assay described herein.

In certain embodiments, an antibody described herein does notcross-react with one or more of HtrA2, HtrA3 and HtrA4.

In certain embodiments, an antibody described herein has a dissociationconstant of ≤500 nM. The dissociation constant may be determined, forexample, by BIAcore using a Fab.

In certain embodiments, an antibody described herein is a monoclonalantibody.

In certain embodiments, an antibody described herein is a human,humanized, or chimeric antibody.

In certain embodiments, an antibody described herein is an antibodyfragment that binds HtrA1.

In certain embodiments, an antibody described herein comprises (a)HVR-H3 comprising the amino acid sequence GTFLTX_(p)WGHYFDY, whereinX_(p) is S or T (SEQ ID NO: 27); (b) HVR-L3 comprising the amino acidsequence QQX_(g)X_(h)X_(i)X_(j)PX_(k)T, wherein X_(g) is S, V or D;X_(h) is Y, D or S; X_(i) is T, S, A, D or N; X_(j) is T, H, N, S, A, Lor R; and X_(k) is P, T, A or S (SEQ ID NO: 24); and (c) HVR-H2comprising the amino acid sequence WIDPYGGDTXoYADSVKG, wherein X_(o) isN or D (SEQ ID NO: 26).

In certain embodiments, an antibody described herein comprises (a)HVR-H3 comprising the amino acid sequence of SEQ ID NO:6; (b) HVR-L3comprising the amino acid sequence of SEQ ID NO:3; and (c) HVR-H2comprising the amino acid sequence of SEQ ID NO:5.

In certain embodiments, an antibody described herein comprises (a)HVR-H3 comprising the amino acid sequence of SEQ ID NO:6; (b) HVR-L3comprising the amino acid sequence of SEQ ID NO:19; and (c) HVR-H2comprising the amino acid sequence of SEQ ID NO:5.

In certain embodiments, an antibody described herein comprises (a)HVR-H3 comprising the amino acid sequence of SEQ ID NO:6; (b) HVR-L3comprising the amino acid sequence of SEQ ID NO:22; and (c) HVR-H2comprising the amino acid sequence of SEQ ID NO:5.

In certain embodiments, an antibody described herein comprises (a)HVR-H1 comprising the amino acid sequence GFX_(l)IX_(m)X_(n)YYIH,wherein X_(l) is N, S or T; X_(m) is S, D, Y or A; and X_(n) is G or D(SEQ ID NO: 25); (b) HVR-H2 comprising the amino acid sequenceWIDPYGGDTX_(o)YADSVKG, wherein X_(o) is N or D (SEQ ID NO:26); and (c)HVR-H3 comprising the amino acid sequence GTFLTX_(p)WGHYFDY, whereinX_(p) is S or T (SEQ ID NO: 27).

In certain embodiments, an antibody described herein further comprises(a) HVR-L1 comprising the amino acid sequenceRASQX_(a)X_(b)X_(c)X_(d)X_(e)X_(f)A, wherein X_(a) is D, S or V; X_(b)is V or I; X_(c) is S, N or G; X_(d) is T or N; X_(e) is A or Y; andX_(f) is V or L (SEQ ID NO: 23); (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2; and (c) HVR-L3 comprising the amino acidsequence QQX_(g)X_(h)X_(i)X_(j)PX_(k)T, wherein X_(g) is S, V or D;X_(h) is Y, D or S; X_(i) is T, S, A, D or N; X_(j) is T, H, N, S, A, Lor R; and X_(k) is P, T, A or S (SEQ ID NO: 24).

In certain embodiments, an antibody described herein comprises (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:5; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:6.

In certain embodiments, an antibody described herein further comprises(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:1; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:3.

In certain embodiments, an antibody described herein comprises (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:20; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:5; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:6.

In certain embodiments, an antibody described herein further comprises(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:18; (b)HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:19.

In certain embodiments, an antibody described herein further comprises(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21; (b)HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:22.

In certain embodiments, an antibody described herein comprises (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:1; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:3.

In certain embodiments, an antibody described herein comprises (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:18; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:19.

In certain embodiments, an antibody described herein comprises (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:21; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:22.

In certain embodiments, an antibody described herein further comprises aheavy chain variable domain framework (FR2) sequence of SEQ ID NO:17.

In certain embodiments, an antibody described herein comprises (a) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:8; (b) a VL sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:7; or (c) a VH sequenceas in (a) and a VL sequence as in (b).

In certain embodiments, an antibody described herein comprises (a) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:29; (b) a VL sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:7; or (c) a VH sequenceas in (a) and a VL sequence as in (b).

In certain embodiments, an antibody described herein comprises (a) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:29; (b) a VL sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO:28; or (c) a VHsequence as in (a) and a VL sequence as in (b).

In certain embodiments, an antibody described herein comprises (a) a VHsequence having at least 95% sequence identity to the amino acidsequence of SEQ ID NO:29; (b) a VI, sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:30; or (c) aVH sequence as in (a) and a VL sequence as in (b).

In certain embodiments, an antibody described herein comprises (a) a VHsequence comprising SEQ ID NO: 32; (b) a VL sequence comprising SEQ IDNO: 31; or (c) a VH sequence comprising SEQ ID NO: 32 and a VL sequencecomprising SEQ ID NO: 31.

In certain embodiments, an antibody described herein comprises a VHsequence of SEQ ID NO:8.

In certain embodiments, an antibody described herein comprises a VLsequence of SEQ ID NO:7.

In certain embodiments, an antibody described herein comprises a VHsequence of SEQ ID NO:30.

In certain embodiments, an antibody described herein comprises a VLsequence of SEQ ID NO:28.

In certain embodiments, an antibody described herein comprises a VLsequence of SEQ ID NO:29.

In certain embodiments, an antibody described herein comprises a VHsequence of SEQ ID NO:8 and a VL sequence of SEQ ID NO:7.

In certain embodiments, an antibody described herein comprises a VHsequence of SEQ ID NO:30 and a VL sequence of SEQ ID NO:28.

In certain embodiments, an antibody described herein comprises a VHsequence of SEQ ID NO:30 and a VL sequence of SEQ ID NO:29.

In certain embodiments, an antibody described herein comprises (a)HVR-H1 comprising the amino acid sequence GFX_(i)IX_(m)X_(n)YYIH,wherein X_(l) is N, S or T; X_(m) is S, D, Y or A; and X_(n) is G or D(SEQ ID NO: 25); (b) HVR-H2 comprising the amino acid sequenceWIDPYGGDTX_(o)YADSVKG, wherein X_(o) is N or D (SEQ ID NO:26); (c)HVR-H3 comprising the amino acid sequence GTFLTX_(p)WGHYFDY, whereinX_(p) is S or T (SEQ ID NO: 27); (d) HVR-L1 comprising the amino acidsequence RASQX_(a)X_(b)X_(c)X_(d)X_(e)X_(f)A, wherein X_(a) is D, S orV; X_(b) is V or I; X_(c) is S, N or G; X_(d) is T or N; X_(e) is A orY; and X_(f) is V or L (SEQ ID NO: 23); (e) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:2; and (f) HVR-L3 comprising the amino acidsequence QQX_(g)X_(h)X_(i)X_(j)PX_(k)T, wherein X_(g) is S, V or D;X_(h) is Y, D or S; X_(i) is T, S, A, D or N; X_(j) is T, H, N, S, A, Lor R; and X_(k) is P, T, A or S (SEQ ID NO: 24).

In certain embodiments, an antibody described herein comprises (a)HVR-H1 comprising an amino acid sequence selected from: SEQ ID NO:4, 20,and 47-51; (b) HVR-H2 comprising an amino acid sequence selected from:SEQ ID NO:5 and 52; (c) HVR-H3 comprising an amino acid sequenceselected from: SEQ ID NO:6 and 53; (d) HVR-L1 comprising an amino acidsequence selected from: SEQ ID NO:1, 18, 21 and 33; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:2; and (f) HVR-L3comprising an amino acid sequence selected from: SEQ ID NO:3, 19, 22,and 34-46.

In certain embodiments, an antibody described herein comprises (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:5; (c) HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:6; (d) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:1; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2; and (f) HVR-L3 comprising the amino acidsequence of SEQ ID NO:3.

In certain embodiments, an antibody described herein comprises (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:20; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:5; (c) HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:6; (d) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:18; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2; and (f) HVR-L3 comprising the amino acidsequence of SEQ ID NO:19.

In certain embodiments, an antibody described herein comprises (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:20; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:5; (c) HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:6; (d) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:21; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2; and (f) HVR-L3 comprising the amino acidsequence of SEQ ID NO:22.

In certain embodiments, an antibody described herein comprises (a)HVR-H3 comprising the amino acid sequence GTFLTX_(p)WGHY, wherein X_(p)is S or T (SEQ ID NO: 89); (b) HVR-L3 comprising the amino acid sequenceX_(g)X_(h)X_(i)X_(j)PX_(k), wherein X_(g) is S, V or D; X_(h) is Y, D orS; X_(i) is T, S, A, D or N; X_(j) is T, H, N, S, A, L or R; and X_(k)is P, T, A or S (SEQ ID NO: 86); and (c) HVR-H2 comprising the aminoacid sequence WIDPYGGDTX_(o), wherein X_(o) is N or D (SEQ ID NO:88).

In certain embodiments, an antibody described herein comprises (a)HVR-H1 comprising the amino acid sequence GFX_(i)IX_(m)X_(n)YY, whereinX_(i) is N, S or T; X_(m) is S, D, Y or A; and X_(n) is G or D (SEQ IDNO:87); (b) HVR-H2 comprising the amino acid sequence WIDPYGGDTX_(o),wherein X_(o) is N or D (SEQ ID NO:88); and (c) HVR-H3 comprising theamino acid sequence GTFLTX_(p)WGHY, wherein X_(p) is S or T (SEQ ID NO:89).

In certain embodiments, an antibody described herein further comprises(a) HVR-L1 comprising the amino acid sequenceX_(a)X_(b)X_(c)X_(d)X_(e)X_(f)r, wherein X_(a) is D, S or V; X_(b) is Vor I; X_(c) is S, N or G; X_(d) is T or N; X_(e) is A or Y; and X_(f) isV or L (SEQ ID NO: 85); (b) HVR-L2 comprising the amino acid sequence ofSEQ ID NO:58; and (c) HVR-L3 comprising the amino acid sequenceX_(g)X_(h)X_(i)X_(j)PX_(k), wherein X_(g) is S, V or D; X_(h) is Y, D orS; X_(i) is T, S, A, D or N; X_(j) is T, H, N, S, A, L or R; and X_(k)is P, T, A or S (SEQ ID NO: 86).

In certain embodiments, an antibody described herein comprises (a)HVR-H1 comprising the amino acid sequence GFX_(l)IX_(m)X_(n)YY, whereinX_(l) is N, S or T; X_(m) is S, D, Y or A; and X_(n) is G or D (SEQ IDNO:87); (b) HVR-H2 comprising the amino acid sequence WIDPYGGDTX_(o),wherein X_(o) is N or D (SEQ ID NO:88); (c) HVR-H3 comprising the aminoacid sequence GTFLTX_(p)WGHY, wherein X_(p) is S or T (SEQ ID NO: 89);(d) HVR-L1 comprising the amino acid sequenceX_(a)X_(b)X_(c)X_(d)X_(e)X_(f), wherein X_(a) is D, S or V; X_(b) is Vor I; X_(c) is S, N or G; X_(d) is T or N; X_(e) is A or Y; and X_(f) isV or L (SEQ ID NO: 85); (e) HVR-L2 comprising the amino acid sequence ofSEQ ID NO:58; and (f) HVR-L3 comprising the amino acid sequenceX_(g)X_(h)X_(i)X_(j)PX_(k), wherein X_(g) is S, V or D; X_(h) is Y, D orS; X_(i) is T, S, A, D or N; X_(j) is T, H, N, S, A, L or R; and X_(k)is P, T, A or S (SEQ ID NO:86).

In certain embodiments, an antibody described herein comprises (a)HVR-H1 comprising an amino acid sequence selected from: SEQ ID NO:4, 20,47-51, and 75-80; (b) HVR-H2 comprising an amino acid sequence selectedfrom: SEQ ID NO:5, 52, and 81-82; (c) HVR-H3 comprising an amino acidsequence selected from: SEQ ID NO:6, 53 and 83-84; (d) HVR-L1 comprisingan amino acid sequence selected from: SEQ ID NO:1, 18, 21, 33, and54-57; (e) HVR-L2 comprising an amino acid sequence selected from: SEQID NO:2 and 58; and (f) HVR-L3 comprising an amino acid sequenceselected from: SEQ ID NO:3, 19, 22, 34-46, and 59-74.

In certain embodiments, an antibody described herein is a full lengthIgG1 or IgG4 antibody.

In another aspect, an isolated nucleic acid encoding an anti-HtrA1antibody described herein is provided.

In another aspect, a host cell comprising an isolated nucleic acidencoding an anti-HtrA1 antibody described herein is provided.

In another aspect, a method of producing an antibody is provided. Themethod may comprise culturing a host cell comprising an isolated nucleicacid encoding an anti-HtrA1 antibody under conditions suitable forexpression of the nucleic acid encoding the anti-HtrA1 antibody. Themethod may further comprise recovering the anti-HtrA1 antibody from thehost cell culture, purifying the anti-HtrA1 antibody, or formulating theanti-HtrA1 antibody with a pharmaceutically acceptable excipient.

In another aspect, an immunoconjugate comprising an anti-HtrA1 antibodyand a cytotoxic agent is provided.

In another aspect, a pharmaceutical formulation comprising an anti-HtrA1antibody and a pharmaceutically acceptable carrier is provided.

In another aspect, the application provides an anti-HtrA1 antibody foruse as a medicament, e.g., for use in treating age-related maculardegeneration (wet or dry), geographic atrophy, diabetic retinopathy,retinopathy of prematurity, or polypoidal choroidal vasculopathy, forinhibiting degeneration of retinal or photoreceptor cells, or forinhibiting HtrA1 protease activity in an eye.

In another aspect, the application provides use of an anti-HtrA1antibody for the manufacture of a medicament, e.g., a medicament for thetreatment of age-related macular degeneration (AMD, wet or dry),geographic atrophy (GA), diabetic retinopathy (DR), retinopathy ofprematurity (ROP), or polypoidal choroidal vasculopathy (PCV), forinhibiting degeneration of retinal or photoreceptor cells, or forinhibiting HtrA1 protease activity in an eye.

In another aspect, the application provides a method of treating anindividual having age-related macular degeneration (wet or dry),geographic atrophy, diabetic retinopathy, retinopathy of prematurity, orpolypoidal choroidal vasculopathy, comprising administering to theindividual an effective amount of an anti-HtrA1 antibody as describedherein.

In another aspect, the application provides a method for inhibitingretinal or photoreceptor cell degeneration in an individual comprisingadministering to the individual an effective amount of an anti-HtrA1antibody as described herein to inhibit retinal or photoreceptor celldegeneration.

In another aspect, the application provides a method of inhibiting HtrA1serine protease activity in an eye of an individual comprisingadministering to the individual an effective amount of an anti-HtrA1antibody as described herein to inhibit HtrA1 serine protease activityin the eye.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B. FIG. 1A shows the light chain variable domain sequence ofanti-HtrA1 antibody94 (YW505.94) (SEQ ID NO:7). FIG. 1B shows the heavychain variable domain sequence of anti-HtrA1 antibody94 (YW505.94) (SEQID NO:8). The residues are numbered according to the Kabat numberingsystem (Kabat. E A., et al., 1991, In: Sequences of proteins ofimmunological interest, fifth edition. National Institutes of Health,Bethesda, Md.). The YW505.94 light chain variable domain sequence isaligned with human KappaI light chain consensus sequence (SEQ ID NO: 9)and the YW505.94 heavy chain variable domain sequence is aligned withthe human subgroup III heavy chain sequence (SEQ ID NO: 10). The boxedsequences are CDRs according to Kabat definitions. The sequencedifferences between anti-HtrA1 (YW505.94) and consensus sequences areshaded.

FIG. 2. Screening of panel of 13 phage derived antibodies (IgG). Singleconcentrations (0.08-0.28 mg/ml final) of IgG were incubated withHuHtrA1 or HuHtrA1_PD and enzyme activity measured in the FRET assay. Ofthe 13 antibodies tested, antibodies YW503.57, YW504.57, YW504.61 andYW505.94 (also referred to as Ab94 or IgG94) strongly inhibited bothHuHtrA1 and HuHtrA1 PD activities.

FIG. 3. Inhibition of HuHtrA1 by IgG94 and Fab94. HuHtrA1 was incubatedwith IgG94 and Fab94 for 20 min at 37° C. Enzyme activity towards thepeptide substrate H2-Opt was measured and fractional activities(v_(i)/v_(o)) calculated from the determined linear velocities. IgG94 at300 nM did not inhibit H2-Opt hydrolysis by trypsin (1 nM) or elastase(1 nM).

FIG. 4. IgG94 inhibits hydrolysis of fluorescent dye-labeled casein(BODIPY FL) by HuHtrA1 and MuHtrA1. HuHtrA1 and MuHtrA1 were incubatedwith IgG94 for 15 min at 37° C. Hydrolysis of casein BODIPY FL reagentwas measured on a microplate reader at 37° C. and the linear rates offluorescence increase determined and expressed as percent of uninhibitedrates (% of control).

FIG. 5. Specificity of IgG94. MuHtrA1_PD, MuHtrA3_PD and MuHtrA4_PD wereincubated with increasing concentrations of IgG94 and enzyme activitiestowards the peptide substrate H2-Opt (top panel) or casein BODIPY FLreagent (bottom panel) determined and expressed as percentage ofuninhibited enzyme activities (% of control)

FIG. 6. Inhibition of HuHtrA1-mediated macromolecular substrate cleavageby IgG94. Increasing concentrations of IgG94 (2.3-150 nM for β-casein;2-125 nM for decorin; 2.3-150 nM for biglycan) were incubated withHuHtrA1 (10 nM for β-casein, 125 nM for decorin, 75 nM for biglycan) for15 min at 37° C. The substrates β-casein, decorin and biglycan (50μg/ml) were added and incubated for 2-14 h. After addition of SDS-samplebuffer the digests were analyzed by SDS-PAGE (non-reducing conditions)and stained by SimplyBlue Safestain.

FIGS. 7A-C. Mapping the functional epitope of IgG94 on HuHtrA1_PD. FIG.7A shows the results of an ELISA measuring the binding of IgG94 toHuHtrA1_PD mutants with alanine substitutions of residues surroundingthe active site. The shaded rows indicate alanine substitutions thatresulted in a greater than 5-fold decrease in binding. FIG. 7B shows thestructure of HuHtrA1_PD (PDB 3NWU) (Clausen, T., et al., Nat Rev MolCell Biol. 12:152-62 (2011)) with Mutated residues indicated. Mediumgray shading shows mutations without loss of IgG94 binding in initialexperiments; dark gray shading shows a subset of residues with >5-foldloss in binding affinity. The three monomers forming the protease domaintrimer, shown in surface representation, are shaded light gray.Catalytic triad residues D250 and S328 are underlined. FIG. 7C shows aclose-up view of the loops harboring the epitope residues N224 and K248on monomer (light gray, as cartoon) of HuHtrA1_PD (PDB 3NWU). Alsoincluded is the catalytic residue H220.

FIGS. 8A-B. SEC-MALLS (size exclusion chromatography—multi-angle laserlight scattering) of the Fab94:HuHtrA1_PD(S328A) complex (FIG. 8A) andthe IgG94:HuHtrA1_PD(S328A) complex (FIG. 8B). Shown are the elutionpeaks by SEC (x-axis) and the masses of individual proteins andcomplexes (y-axis; dotted lines across the elution peaks).

FIG. 9. Hypothetical ‘cage model’ of the IgG:HuHtrA1-PD complex.

FIGS. 10A-B. Alignment of human HtrA1 (SEQ ID NO:13), murine HtrA1 (SEQID NO:14), murine HtrA3 (SEQ ID NO:15), and murine HtrA4 (SEQ ID NO:16).

FIG. 11. HtrA1 mRNA expression increases in the retina in a mouse modelof constant light exposure (top left panel). HtrA2 levels do not varysignificantly in the same model (top right panel). HtrA1 expression issignificantly higher than HtrA2, HtrA3 and HtrA4 expression levels inthe mouse retina at baseline (bottom panel).

FIG. 12. Increased bi-polar cell/Muller cell responses in the absence ofHtrA1 expression in a mouse model of constant light exposure.

FIG. 13. Sparing of retina in the absence of HtrA1 expression in a mousemodel of constant light exposure.

FIG. 14. Sparing of outer nuclear layer (ONL) photoreceptor cells in theabsence of HtrA1 expression in a mouse model of constant light exposure.

FIG. 15. Light chain HVR sequences for affinity-improved variants ofanti-HtrA1 antibody YW505.94a.

FIG. 16. Heavy chain HVR sequences for affinity-improved variants ofanti-HtrA1 antibody YW505.94a.

FIG. 17. Results of a phage competition assay demonstrating the bindingof YW505.94a affinity-improved variants against HuHtrA1.

FIG. 18. Results of a phage competition assay demonstrating the bindingof YW505.94a affinity-improved variants against MuHtrA1.

FIGS. 19A-C. Results of ELISA assay showing levels of HtrA1 protein inrat tissues (FIG. 19A), mouse eye fluid (FIG. 19B) and mouse retinatissue (FIG. 19C).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-HtrA1 antibody” and “an antibody that binds to HtrA1”refer to an antibody that is capable of binding HtrA1 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting HtrA1. In one embodiment, the extent ofbinding of an anti-HtrA1 antibody to an unrelated, non-HtrA1 protein isless than about 10% of the binding of the antibody to HtrA1 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to HtrA1 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM,≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less,e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainembodiments, an anti-HtrA1 antibody binds to an epitope of HtrA1 that isconserved among HtrA1 from different species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework region” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1 (L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition. AnHVR region as used herein comprise any number of residues located withinpositions 24-36 (for L1), 46-56 (for L2), 89-97 (for L3), 26-35B (forH1), 47-65 (for H2), and 93-102 (for H3).

Therefore, an HVR includes residues in positions described previously:

A) 24-34 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987);

B) 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and95-102 of H3 (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991).

C) 30-36 (L1), 46-55 (L2), 89-96 (L3), 30-35 (H1), 47-58 (H2), 93-100a-j(H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996).

With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. CDRs also comprise“specificity determining residues,” or “SDRs,” which are residues thatcontact antigen. SDRs are contained within regions of the CDRs calledabbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2,a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633(2008).) Unless otherwise indicated, HVR residues and other residues inthe variable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally, at a chromosomal location thatis different from its natural chromosomal location, or contains onlycoding sequences.

“Isolated nucleic acid encoding an anti-HtrA1 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however. % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “High-temperature requirement associated A” or “HtrA1,” as usedherein, refers to any native HtrA1 from any vertebrate source, includingmammals such as primates (e.g. humans) and rodents (e.g., mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed HtrA1 as well as any form of HtrA1 that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of HtrA1, e.g., splice variants or allelic variants. The aminoacid sequence of an exemplary HtrA1 is shown in SEQ ID NO:13. Exemplaryfragments of human HtrA1 include fragments comprising, consistingessentially of, or consisting of amino acids Q23-P480 or P161-K379.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

II. Compositions and Methods

In one aspect, the invention is based, in part, on the discovery that areduction of HtrA1 activity has protective effects on photoreceptorcells in the eye, the outer nuclear layer, and on elecroretinogramfunctionality. In certain embodiments, antibodies that bind to HtrA1 areprovided. Antibodies of the invention are useful, e.g., for thediagnosis or treatment of various diseases associated with HtrA1activity, including ocular disorders such as age-related maculardegeneration or geographic atrophy.

A. Exemplary Anti-HtrA1 Antibodies

In one aspect, the invention provides isolated antibodies that bind toHtrA1. In certain embodiments, an anti-HtrA1 antibody has one or more ofthe following properties: (i) has an IC₅₀ of less than 50 nM, 30 nM, 25nM, 20 nM, 15 nM, 10 nM, 5 nM, 3 nM, 2.5 nM, 2 nM, 1 nM, or less for oneor more HtrA1 substrates; (ii) binds to HtrA1 with a ratio of 1 variabledomain to one subunit of an HtrA1 trimer (e.g., a Fab binds to an HtrA1trimer with a ratio of 3 Fab to 1 HtrA1 trimer, and an IgG binds to anHtrA1 trimer with a ratio of 3 IgG to 2 HtrA1 trimers), (iii) forantibodies comprising two variable domains, binds to HtrA1 in a mannerthat results in the forming a “cage” similar to that shown in FIG. 9,(iv) does not prevent trimer formation of HtrA1, (v) binds to one ormore residues in Loop C of the HtrA1 protein, (vi) binds to the proteasedomain of HtrA1, (vii) binds to an epitope comprising one or both ofamino acids N224 or K248 of SEQ ID NO:13, or amino acids equivalentthereto in a different HtrA1 sequence (e.g., amino acids N224 and K248of SEQ ID NO:14, see FIGS. 10A-B); (viii) binds to an epitope comprisingone or more of residues N224, K248, V201, T223, K243, K225, E247 andH220 of SEQ ID NO:13, or amino acids equivalent thereto in a differentHtrA1 sequence; (ix) cross-reacts with murine HtrA1; (x) does notcross-react with HtrA2, HtrA3 and/or HtrA4; (xi) binds to HtrA1competitively with an antibody comprising a VH sequence of SEQ ID NO:8and a VL sequence of SEQ ID NO:7, or (xii) binds to HtrA1 with adissociation constant of ≤500 nM, or (xiii) inhibits complex formationbetween HtrA1 and α1-antitrypsin (A1AT).

In one aspect, the invention provides an anti-HtrA1 antibody comprisingat least one, two, three, four, five, or six HVRs selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:87; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:88; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:89; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:85; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:58; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:86. In one embodiment,the invention provides an anti-HtrA1 antibody comprising at least one,two, three, four, five, or six HVRs selected from (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:25; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:26; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:27; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:23; (e) HVR-L2 comprising the amino acid sequence of SEQ IDNO:2; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:24.In one embodiment, the invention provides an anti-HtrA1 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising an amino acid sequence selected from: SEQ IDNO:4, 20, 47-51 and 75-80; (b) HVR-H2 comprising an amino acid sequenceselected from: SEQ ID NO:5, 52 and 81-82; (c) HVR-H3 comprising an aminoacid sequence selected from: SEQ ID NO:6, 53 and 83-84; (d) HVR-L1comprising an amino acid sequence selected from: SEQ ID NO:1, 18, 21, 33and 54-57; (e) HVR-L2 comprising an amino acid sequence selected from:SEQ ID NO:2 and 58; and (f) HVR-L3 comprising an amino acid sequenceselected from: SEQ ID NO:3, 19, 22, 34-46 and 59-74. In one embodiment,the invention provides an anti-HtrA1 antibody comprising at least one,two, three, four, five, or six HVRs selected from (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:4; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:5; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:6; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:1; (e) HVR-L2 comprising the amino acid sequence of SEQ IDNO:2; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:3.In one embodiment, the invention provides an anti-HtrA1 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:20; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:6; (d) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:18; (e) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:2; and (f) HVR-L3 comprising the amino acidsequence of SEQ ID NO:19. In one embodiment, the invention provides ananti-HtrA1 antibody comprising at least one, two, three, four, five, orsix HVRs selected from (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:20; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:21; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:2; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:22.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:87; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:88; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:89. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:89. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:89 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:86. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:89, HVR-L3 comprising the amino acid sequence of SEQ ID NO:86, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:88. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:87; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:88; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:89.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:25; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:26; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:27. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:27 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:24. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:27, HVR-L3 comprising the amino acid sequence of SEQ ID NO:24, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:26. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:25; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:26; and (c) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:27.

In one embodiment, the invention provides an antibody comprising atleast one, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising an amino acid sequence selected from: SEQ ID NO:4, 20,47-51 and 75-80; (b) HVR-H2 comprising an amino acid sequence selectedfrom SEQ ID NO:5, 52 and 81-82; and (c) HVR-H3 comprising an amino acidsequence selected from: SEQ ID NO:6, 53 and 83-84. In one embodiment,the antibody comprises HVR-H3 comprising an amino acid sequence selectedfrom SEQ ID NO:6 and 53. In another embodiment, the antibody comprisesHVR-H3 comprising an amino acid sequence selected from SEQ ID NO:6, 53and 83-84 and HVR-L3 comprising an amino acid sequence selected from SEQID NO:3, 19, 22, 34-46 and 59-74. In a further embodiment, the antibodycomprises HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:6, 53 and 83-84, HVR-L3 comprising an amino acid sequence selectedfrom SEQ ID NO: 3, 19, 22, 34-46 and 59-74, and HVR-H2 comprising anamino acid sequence selected from SEQ ID NO:5, 52 and 81-82. In afurther embodiment, the antibody comprises (a) HVR-H1 comprising anamino acid sequence selected from SEQ ID NO:4, 20, 47-51 and 75-80; (b)HVR-H2 comprising an amino acid sequence selected from SEQ ID NO:5, 52and 81-82; and (c) HVR-H3 comprising an amino acid sequence selectedfrom SEQ ID NO:6, 53 and 83-84.

In one embodiment, the invention provides an antibody comprising atleast one, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:5; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:6. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:6. In another embodiment, the antibody comprises HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:6 and HVR-L3 comprising the aminoacid sequence of SEQ ID NO:3. In a further embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:6,HVR-L3 comprising the amino acid sequence of SEQ ID NO:3, and HVR-H2comprising the amino acid sequence of SEQ ID NO:5. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:4; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:5; and (c) HVR-H3 comprising the amino acid sequence of SEQID NO:6.

In one embodiment, the invention provides an antibody comprising atleast one, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:20; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:5; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:6. In one embodiment,the antibody comprises HVR-H3 comprising the amino acid sequence of SEQID NO:20. In another embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:20 and HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:19. In a further embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO:20, HVR-L3 comprising the amino acid sequence of SEQ ID NO:19, andHVR-H2 comprising the amino acid sequence of SEQ ID NO:5. In anotherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO:20 and HVR-L3 comprising the amino acid sequenceof SEQ ID NO:22. In a further embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:20, HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:22, and HVR-H2 comprising the aminoacid sequence of SEQ ID NO:5. In a further embodiment, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:20;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:5; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:6.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:85; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:58; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:86. In one embodiment,the invention provides an antibody comprising at least one, at leasttwo, or all three VL HVR sequences selected from (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:23; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:2; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:24. In one embodiment, the antibody comprises (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:85; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:58; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:86. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:23; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:24.

In another embodiment, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising an amino acid sequence selected from SEQ ID NO:1, 18,21, 33 and 54-57; (b) HVR-L2 comprising an amino acid sequence selectedfrom SEQ ID NO:2 and 58; and (c) HVR-L3 comprising an amino acidsequence selected from SEQ ID NO:3, 19, 22, 34-46 and 59-74. In oneembodiment, the antibody comprises (a) HVR-L1 comprising an amino acidsequence selected from SEQ ID NO:1, 18, 21, 33 and 54-57; (b) HVR-L2comprising an amino acid sequence selected from SEQ ID NO:2 and 58; and(c) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:3,19, 22, 34-46 and 59-74.

In another embodiment, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:1; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:3. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:1; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:3.

In another embodiment, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:18; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:19. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:18; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:19.

In another embodiment, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:21; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:22. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:21; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:22.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:87, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:88, and (iii) HVR-H3 comprising the amino acid sequence of SEQ IDNO:89; and (b) a VL domain comprising at least one, at least two, or allthree VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:85, (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:58, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:86. In one embodiment, an antibody of theinvention comprises (a) a VH domain comprising at least one, at leasttwo, or all three VH HVR sequences selected from (i) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:25, (ii) HVR-H2 comprising theamino acid sequence of SEQ ID NO:26, and (iii) HVR-H3 comprising theamino acid sequence of SEQ ID NO:27; and (b) a VL domain comprising atleast one, at least two, or all three VL HVR sequences selected from (i)HVR-L1 comprising the amino acid sequence of SEQ ID NO:23, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:2, and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:24.

In another embodiment, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising an amino acid sequenceselected from SEQ ID NO:4, 20, 47-51 and 75-80, (ii) HVR-H2 comprisingan amino acid sequence selected from SEQ ID NO:5, 52 and 81-82, and(iii) HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:6, 53 and 83-84; and (b) a VL domain comprising at least one, atleast two, or all three VL HVR sequences selected from (i) HVR-L1comprising an amino acid sequence selected from SEQ ID NO:1, 18, 21, 33and 54-57, (ii) HVR-L2 comprising an amino acid sequence selected fromSEQ ID NO:2 and 58, and (c) HVR-L3 comprising an amino acid sequenceselected from SEQ ID NO:3, 19, 22, 34-46 and 59-74.

In another embodiment, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:4, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:5, and (iii) HVR-H3 comprising the amino acid sequence of SEQ IDNO:6; and (b) a VL domain comprising at least one, at least two, or allthree VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:1, (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:3.

In another embodiment, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:20, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:5, and (iii) HVR-H3 comprising the amino acid sequence of SEQ IDNO:6; and (b) a VL domain comprising at least one, at least two, or allthree VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:18, (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:19.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO:20, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:5, and (iii) HVR-H3 comprising the amino acid sequence of SEQ IDNO:6; and (b) a VL domain comprising at least one, at least two, or allthree VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:21, (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:22.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:87; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:88; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:89; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:85; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:58; and (f) HVR-13comprising the amino acid sequence of SEQ ID NO:86. In one embodiment,the invention provides an antibody comprising (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:25; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:26; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:27; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:23; (e) HVR-L2 comprising the amino acid sequence of SEQ IDNO:2; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:24.

In another embodiment, the invention provides an antibody comprising (a)HVR-H1 comprising an amino acid sequence selected from SEQ ID NO:4, 20,47-51 and 75-80; (b) HVR-H2 comprising an amino acid sequence selectedfrom SEQ ID NO:5, 52 and 81-82; (c) HVR-H3 comprising an amino acidsequence selected from SEQ ID NO:6, 53 and 83-84; (d) HVR-L1 comprisingan amino acid sequence selected from SEQ ID NO:1, 18, 21, 33 and 54-57;(e) HVR-L2 comprising an amino acid sequence selected from SEQ ID NO:2and 58; and (f) HVR-L3 comprising an amino acid sequence selected fromSEQ ID NO:3, 19, 22, 34-46 and 59-74.

In another embodiment the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:5; (c) HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:6; (d) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:1; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2; and (f) HVR-L3 comprising the amino acidsequence of SEQ ID NO:3.

In another embodiment the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:20; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:5; (c) HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:6; (d) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:18; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2; and (f) HVR-L3 comprising the amino acidsequence of SEQ ID NO:19.

In another embodiment the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:20; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:5; (c) HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:6; (d) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:21; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2; and (f) HVR-L3 comprising the amino acidsequence of SEQ ID NO:22.

In any of the above embodiments, an anti-HtrA1 antibody is humanized. Inone embodiment, an anti-HtrA1 antibody comprises HVRs as in any of theabove embodiments, and further comprises an acceptor human framework,e.g. a human immunoglobulin framework or a human consensus framework. Inanother embodiment, an anti-HtrA1 antibody comprises HVRs as in any ofthe above embodiments, and further comprises a VH comprising an FR2sequence of SEQ ID NO:17.

In another aspect, an anti-HtrA1 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of any one of SEQ ID NO:8 or 29. In certain embodiments, a VHsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-HtrA1 antibody comprising that sequence retains the ability to bindto HtrA1. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO:8 or 29. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-HtrA1antibody comprises the VH sequence in SEQ ID NO:8, 29 or 32, includingpost-translational modifications of that sequence. In a particularembodiment, the VH comprises one, two or three HVRs selected from: (a)HVR-H1 comprising an amino acid sequence selected from: SEQ ID NO:4, 20,25 and 47-51, (b) HVR-H2 comprising an amino acid sequence selected fromSEQ ID NO:5, 26 and 52, and (c) HVR-H3 comprising an amino acid sequenceselected from SEQ ID NO:6, 27 and 53.

In another aspect, an anti-HtrA1 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of any one of SEQ ID NO:7, 28 or 30.In certain embodiments, a VL sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions(e.g., conservative substitutions), insertions, or deletions relative tothe reference sequence, but an anti-HtrA1 antibody comprising thatsequence retains the ability to bind to HtrA1. In certain embodiments, atotal of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO:7, 28 or 30. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-HtrA1 antibody comprisesthe VL sequence in SEQ ID NO:7, 28, 30 or 31, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising an amino acid sequence selected from SEQ ID NO:1, 18,21, 23 and 33; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO:2; and (c) HVR-L3 comprising an amino acid sequence selected from SEQID NO:3, 19, 22, 24 and 34-36.

In another aspect, an anti-HtrA1 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO:32 and SEQ IDNO:31, respectively, including post-translational modifications of thosesequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:8 and SEQ ID NO:7, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:29 and SEQID NO:28, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:29 and SEQ ID NO:30, respectively, includingpost-translational modifications of those sequences.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-HtrA1 antibody provided herein. For example,in certain embodiments, an antibody is provided that binds to the sameepitope as an anti-HtrA1 antibody comprising a VH sequence of SEQ IDNO:8 and a VL sequence of SEQ ID NO:7. In certain embodiments, anantibody is provided that binds to an epitope of HtrA1 containingresidue N224, K248 or both of SEQ ID NO:13, or residues equivalentthereto in a different HtrA1 sequence. In certain embodiments, theepitope of HtrA1 further comprising one or more of the followingresidues V201, T223, K243, K225, E247 and H220 of SEQ ID NO:13, or aminoacids equivalent thereto in a different HtrA1 sequence. In certainembodiments, an antibody is provided that binds to an epitope of HtrA1containing one or more of residues N224, K248 and V201 or all of theforegoing of SEQ ID NO:13, or residues equivalent thereto in a differentHtrA1 sequence. In certain embodiments, an antibody is provided thatbinds to an epitope of HtrA1 containing one or more of residues N224,K248, V201, T223 and K243 or all of the foregoing of SEQ ID NO:13, orresidues equivalent thereto in a different HtrA1 sequence. In certainembodiments, an antibody is provided that binds to an epitope of HtrA1containing one or more of residues N224, K248, V201, T223, K243, K225,E247 and H22A or all of the foregoing of SEQ ID NO:13, or residuesequivalent thereto in a different HtrA1 sequence. In certainembodiments, the epitope is a linear epitope. In other embodiments, theepitope is a conformational epitope.

In a further aspect of the invention, an anti-HtrA1 antibody accordingto any of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-HtrA1antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is a fulllength antibody, e.g., an intact IgG1 antibody or other antibody classor isotype as defined herein.

In certain embodiments, an anti-HtrA1 antibody according to any of theabove embodiments is not an antibody having a VH sequence of SEQ ID NO:8and a VL sequence of SEQ ID NO:7.

In a further aspect, an anti-HtrA1 antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed. e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology, U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Aced. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for HtrA1 and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of HtrA1. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express HtrA1. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to HtrA1 as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “conservative substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr. Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs. e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibody variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fec region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Inmmunol. 18(12): 1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Inmmunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain: A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-HtrA1 antibody described hereinis provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-HtrA1 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-HtrA1 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W 138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982): MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-HtrA1 antibodies provided herein may be identified, screened for,or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes with Fab94 or IgG94 for binding to HtrA1. Incertain embodiments, such a competing antibody binds to the same epitope(e.g., a linear or a conformational epitope) that is bound by Fab94 orIgG94. Detailed exemplary methods for mapping an epitope to which anantibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.).

In an exemplary competition assay, immobilized HtrA1 is incubated in asolution comprising a first labeled antibody that binds to HtrA1 (e.g.,Fab94 or IgG94) and a second unlabeled antibody that is being tested forits ability to compete with the first antibody for binding to HtrA1. Thesecond antibody may be present in a hybridoma supernatant. As a control,immobilized HtrA1 is incubated in a solution comprising the firstlabeled antibody but not the second unlabeled antibody. After incubationunder conditions permissive for binding of the first antibody to HtrA1,excess unbound antibody is removed, and the amount of label associatedwith immobilized HtrA1 is measured. If the amount of label associatedwith immobilized HtrA1 is substantially reduced in the test samplerelative to the control sample, then that indicates that the secondantibody is competing with the first antibody for binding to HtrA1. SeeHarlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-HtrA1 antibodiesthereof having biological activity. Biological activity may include,e.g., blocking, antagonizing, suppressing, interfering, modulatingand/or reducing one or more biological activities of HtrA1. Antibodieshaving such biological activity in vivo and/or in vitro are alsoprovided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity. In certain embodiments, an anti-HtrA1 antibodybinds to HtrA1 and reduces or inhibits its serine protease activity forone or more HtrA1 substrates, including, for example, the H2-Optpeptide, β-casein or BODIPY FL casein substrates as described in theExamples below, or any other suitable HtrA1 substrate. In certainembodiments, an anti-HtrA1 antibody inhibits HtrA1 serine proteaseactivity with an IC₅₀ of less than 50 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10nM, 5 nM, 3 nM, 2.5 nM, 2 nM, 1 nM, or less for one or more HtrA1substrates. In certain embodiments, an anti-HtrA1 antibody protectsphotoreceptor cells from degradation, protects the thickness of theouter nuclear layer, or protects electroretinogram functional activityin an ocular disease model, such as the constant light exposure mousemodel described in the Examples below.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-HtrA1antibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC 1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example Tc^(99m) orI¹²³, or a spin label for nuclear magnetic resonance (NMR) imaging (alsoknown as magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology. Inc., Rockford, Ill., U.S.A.).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-HtrA1 antibodies provided hereinis useful for detecting the presence of HtrA1 in a biological sample.The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue, such as a sample comprising photoreceptorcells, retinal pigment epithelium cells, cells of the outer nuclearlayer, the inner nuclear layer, Muller cells, ciliary epithelium, orretinal tissue.

In one embodiment, an anti-HtrA1 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of HtrA1 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-HtrA1 antibody as described herein under conditionspermissive for binding of the anti-HtrA1 antibody to HtrA1, anddetecting whether a complex is formed between the anti-HtrA1 antibodyand HtrA1. Such method may be an in vitro or in vivo method. In oneembodiment, an anti-HtrA1 antibody is used to select subjects eligiblefor therapy with an anti-HtrA1 antibody, e.g. where HtrA1 is a biomarkerfor selection of patients.

In certain embodiments, a patient suitable for treatment with ananti-HtrA1 antibody may be identified by detecting one or morepolymorphisms in the HtrA1 gene or HtrA1 control sequence, such as theHtrA1 promoter polymorphism rs11200638(G/A) (see e.g., A. DeWan, et al.,Science 314: 989-992 (2006)).

Exemplary disorders that may be diagnosed using an antibody of theinvention include ocular disorders, such as, for example, wetage-related macular degeneration (AMD), dry age-related maculardegeneration, geographic atrophy (GA), diabetic retinopathy (DA),retinopathy of prematurity (ROP), or polypoidal choroidal vasculopathy(PCV).

In certain embodiments, labeled anti-HtrA1 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, j-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-HtrA1 antibody as describedherein are prepared by mixing such antibody having the desired degree ofpurity with one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, for treating an ocular disorder associated undesiredneovascularization, such as wet AMD, it may be desirable to furtherprovide an anti-angiogenic therapy, such as an anti-VEGF therapy likeLUCENTIS™ (ranibizumab). Such active ingredients are suitably present incombination in amounts that are effective for the purpose intended. Inother embodiments, treatment of a disease or disorder associatedundesirable ocular neovascularization may involve a combination of ananti-HtrA1 antibody and photodynamic therapy (e.g., with MACUGEN™ orVISUDYNE™).

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-HtrA1 antibodies provided herein may be used intherapeutic methods.

In certain embodiments, an anti-HtrA1 antibody is useful for inhibitingthe degeneration of retinal cells (such as, for example, retinal pigmentepithelium (RPE) cells) or photoreceptor cells in an eye of a patientsuffering from or at risk for developing an ocular disorder (e.g., apatient having an HtrA1 polymorphism, increased HtrA1 expression in theeye, or drusen in at least one eye). In certain embodiments, ananti-HtrA1 antibody may be used for slowing the progression to latestage geographic atrophy or dry age-related macular degeneration in aneye of a patient having drusen in the eye to be treated. In certainembodiments, a patient suitable for treatment with an anti-HtrA1antibody may be identified by detecting the onset of disease in at leastone eye of the patient. For example, certain patients may be selected bydetecting drusen, geographic atrophy, or choroidal neovascularization inone eye, while the other eye is symptom free. Such patients may be goodcandidates for preventative treatment in the symptom free eye, e.g., todelay the onset or reduce the severity of such symptoms (e.g., drusen,geographic atrophy, and/or choroidal neovascularization) in the symptomfree eye. Alternatively, the symptomatic eye may be treated, or both thesymptomatic and symptom free eye may be treated in accordance with themethods described herein. Accordingly, an anti-HtrA1 antibody may beused for preventing or inhibiting the progression of an ocular disorderin an eye of patient, wherein the patient has developed drusen, wet AMD,or geographic atrophy in the other eye, but the eye being treated is notyet symptomatic. Alternatively, the symptomatic eye is treated, or boththe symptomatic and the symptom free eye are treated.

In another embodiment, an anti-HtrA1 antibody is useful for treatingarthritis.

In one aspect, an anti-HtrA1 antibody for use as a medicament isprovided. In further aspects, an anti-HtrA1 antibody for use in treatingan ocular disease or disorder, such as, for example, AMD (wet or dry),GA, DR, PCV or ROP, is provided. In certain embodiments, an anti-HtrA1antibody for use in a method of treatment is provided. In certainembodiments, the invention provides an anti-HtrA1 antibody for use in amethod of treating an individual having an ocular disease or disordercomprising administering to the individual an effective amount of theanti-HtrA1 antibody. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below. Infurther embodiments, the invention provides an anti-HtrA1 antibody foruse in inhibiting degeneration of retinal cells (such as, for example,RPE cells) or photoreceptor cells in an eye of a patient. In certainembodiments, the invention provides an anti-HtrA1 antibody for use in amethod of inhibiting degeneration of retinal or photoreceptor cells inan individual comprising administering to the individual an effectiveamount of the anti-HtrA1 antibody to inhibit degeneration of retinal orphotoreceptor cells. In further embodiments, the invention provides ananti-HtrA1 antibody for use in inhibiting HtrA1 serine protease activityin an eye of a patient. In certain embodiments, the invention providesan anti-HtrA1 antibody for use in a method of inhibiting HtrA1 serineprotease activity in an eye of an individual comprising administering tothe individual an effective amount of the anti-HtrA1 antibody to inhibitHtrA1 serine protease activity in the eye. An “individual” according toany of the above embodiments is preferably a human. In furtherembodiments, the invention provides an anti-HtrA1 antibody for use ininhibiting PCV in a patient in need thereof, e.g., a patient havingchoroidal vascular networks with polyp-like aneurysmal dilations.

In a further aspect, the invention provides for the use of an anti-HtrA1antibody in the manufacture or preparation of a medicament. In oneembodiment, the medicament is for treatment of an ocular disorder, suchas, for example, AMD (wet or dry), GA. DR, PCV or ROP. In a furtherembodiment, the medicament is for use in a method of treating an oculardisorder comprising administering to an individual having the oculardisorder an effective amount of the medicament. In one such embodiment,the method further comprises administering to the individual aneffective amount of at least one additional therapeutic agent, e.g., asdescribed below. In a further embodiment, the medicament is forinhibiting the degradation of retinal cells or photoreceptor cells in aneye of an individual. In a further embodiment, the medicament is for usein a method of inhibiting degeneration of retinal cells or photoreceptorcells in an individual comprising administering to the individual anamount effective of the medicament to inhibit degeneration of retinal orphotoreceptor cells in the individual. In a further embodiment, themedicament is for inhibiting HtrA1 1 serine protease activity in the eyeof an individual. In a further embodiment, the medicament is for use ina method of inhibiting HtrA1 serine protease activity in an eye of anindividual comprising administering to the individual an amounteffective of the medicament to inhibit HtrA1 serine protease activity inthe eye. An “individual” according to any of the above embodiments maybe a human.

In a further aspect, the invention provides a method for treating anocular disorder, such as, for example, AMD (wet or dry), GA, DR, PCV orROP. In one embodiment, the method comprises administering to anindividual having such an ocular disorder an effective amount of ananti-HtrA1 antibody. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, as described below. An“individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for inhibitingdegeneration of retinal or photoreceptor cells in an individual. In oneembodiment, the method comprises administering to the individual aneffective amount of an anti-HtrA1 antibody to inhibit degeneration ofretinal or photoreceptor cells in the individual. In one embodiment, an“individual” is a human.

In a further aspect, the invention provides a method for inhibitingHtrA1 serine protease activity in an eye of an individual. In oneembodiment, the method comprises administering to the individual aneffective amount of an anti-HtrA1 antibody to inhibit HtrA1 serineprotease activity in an eye of the individual. In one embodiment, an“individual” is a human.

The efficacy of the treatment of an ocular disorder using an anti-HtrA1antibody, can be measured by various endpoints commonly used inevaluating intraocular diseases, such as, for example, performing an eyeexam, measuring intraocular pressure, assessing visual acuity, measuringslitlamp pressure, assessing intraocular inflammation, measuring thesize of CNV, measuring the leakage of CNV (e.g., by Fluoresceinangiography), measuring the amount of drusen, measuring the location ofdrusen, etc. In certain embodiments, vision loss can be assessed, forexample, by measuring the mean change in best correction visual acuity(BCVA) from baseline to a desired time point (e.g., where the BCVA isbased on Early Treatment Diabetic Retinopathy Study (ETDRS) visualacuity chart and assessment at a test distance of 4 meters), measuringthe proportion of subjects who lose fewer than 15 letters in visualacuity at a desired time point compared to baseline, measuring theproportion of subjects who gain greater than or equal to 15 letters invisual acuity at a desired tine point compared to baseline, measuringthe proportion of subjects with a visual-acuity Snellen equivalent of20/2000 or worse at a desired time point, or measuring the NEI VisualFunctioning Questionnaire.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-HtrA1 antibodies provided herein, e.g., foruse in any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the anti-HtrA1 antibodiesprovided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of the anti-HtrA1antibodies provided herein and at least one additional therapeuticagent, e.g., as described below.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is a therapeutic agent suitable for treatment of an oculardisorder associated with undesirable neovascularization in the eye, suchas, for example, wet AMD. Suitable therapeutic agents include, forexample, anti-angiogenic therapies such as an anti-VEGF therapy likeLUCENTIS™ (ranibizumab).

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies of the invention can alsobe used in combination with photodynamic therapy (e.g., with MACUJGEN™or VISUDYNE™).

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In an exemplary embodiment, an antibody ofthe invention (and any additional therapeutic agent) can be administeredby intravitreal injection. Dosing can be by any suitable route, e.g. byinjections, such as intravenous, intravitreal or subcutaneousinjections, depending in part on whether the administration is brief orchronic. Various dosing schedules including but not limited to single ormultiple administrations over various time-points, bolus administration,and pulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-HtrA1 antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-HtrA1 antibody.

SEQUENCE KEY Light chain HVRs for Anti-HtrA1 Antibody YW505.94 HVR L1:(SEQ ID NO: 1) RASQDVSTAVA HVR L2: (SEQ ID NO: 2) SASFLYS HVR L3:(SEQ ID NO: 3) QQSYTTPPTHeavy chain HVRs for Anti-HtrA1 Antibody YW505.94 HVR H1: (SEQ ID NO: 4)GFNISGYYIH HVR H2: (SEQ ID NO: 5) WIDPYGGDTNYADSVKG HVR H3:(SEQ ID NO: 6) GTFLTSWGHYFDYLight chain VR for Anti-HtrA1 Antibody YW505.94 (SEQ ID NO: 7)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKRHeavy chain VR for Anti-HtrA1 Antibody YW505.94 (SEQ ID NO: 8)EVQLVESGGGLVQPGGSLRLSCAASGFNISGYYIHWVRQAPGKGLEWVGWIDPYGGDTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGTFLTSWGHYFDYWGQGTHuman HtrA1 (full length sequence in caps, protease domain is underlined, residues N224 andK248 are shaded) (SEQ ID NO: 13)mqipraallpllllllaapasaQLSRAGRSAPLAAGCPDRCEPARCPPQPEHCEGGRARDACGCCEVCGAPEGAACGLQEGPCGEGLQCVVPFGVPASATVRRRAQAGLCVCASSEPVCGSDANTYANLCQLRAASRRSERLHRPPVIVLQRGACGQGQEDPNSLRHKYNFIADVVEKIAPAVVHIEL

ADIALIKIDHQGKLPVLLLGRSSELRPGEFVVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRNSDMDYIQTDAIINYGNSGGPLVNLDGEVIGINTLKVTAGISFAIPSDKIKKFLTESHDRQAKGKAITKKKYIGIRMMSLTSSKAKELKDRHRDFPDVISGAYIIEVIPDTPAEAGGLKENDVIISINGQSVVSANDVSDVIKRESTLNMVVRRGNEDIMITVIPEEIDIPMurine HtrA1 (full length sequence in caps, protease domain is underlined)(SEQ ID NO: 14)mqslrttllsllllllaapslalPSGTGRSAPAATVCPEHCDPTRCAPPPTDCEGGRVRDACGCCEVCGALEGAACGLQEGPCGEGLQCVVPFGVPASATVRRRAQAGLCVCASSEPVCGSDAKTYTNLCQLRAASRRSEKLRQPPVIVLQRGACGQGQEDPNSLRHKYNFIADVVEKIAPAVVHIELYRKLPFSKREVPVASGSGFIVSEDGLIVTNAHVVTNKNRVKVELKNGATYEAKIKDVDEKADIALIKIDHQGKLPVLLLGRSSELRPGEFVVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRNSDMDYIQTDAIINYGNSGGPLVNLDGEVIGINTLKVTAGISFAIPSDKIKKFLTESHDRQAKGKAVTKKKYIGIRMMSLTSSKAKELKDRHRDFPDVLSGAYIIEVIPDTPAEAGGLKENDVIISINGQSVVTANDVSDVIKKENTLNMVVRRGNEDIVITVIPEEIDPMurine HtrA3 (protease domain is underlined) (SEQ ID NO: 15)MQARALLPATLAILATLAVLALAREPPAAPCPARCDVSRCPSPRCPGGYVPDLCNCCLVCAASEGEPCGRPLDSPCGDSLECVRGVCRCRWTHTVCGTDGHTYADVCALQAASRRALQVSGTPVRQLQKGACPSGLHQLTSPRYKFNFIADVVEDIAPAVVHIELFLRHPLFGRNVPLSSGSGFIMSEAGLIVTNAHVVSSSSTASGRQQLKVQLQNGDAYEATIQDIDKKSDIATIVIHPKKKLPVLLLGHSADLRPGEFVVAIGSPFALQNTVTTGIVSTAQRDGKELGLRDSDMDYIQTDAIINYGNSGGPLVNLDGEVIGINTLKVAAGISFAIPSDRITRFLSEFQNKHVKDWKKRFIGIRMRTITPSLVEELKAANPDFPAVSSGIYVQEVVPNSPSQRGGIQDGDIIVKVNGRPLADSSELQEAVLNESSLLLEVRRGNDDLLFSIIPEVVMMurine HtrA4 (protease domain is underlined) (SEQ ID NO: 16)MSFQRLWAVRTQFLLLWLLLPAVPVPWAEARRSRVSLPCPDACDPTRCPTLPTCSAGLAPVPDRCGCCRVCAAAEGQECGGARGRPCAPRLRCGAPFSRDPSGGAWLGTCGCAEGAEDAVVCGSDGRTYPSLCALRKENRAARQRGALPAVPVQKGACEEAGTTRAGRLRRKYNFIAAVVEKVAPSVVHLQLFRRSPLTNQEIPSSSGSGFIVSEDGLIVTNAHVLTNQQKIQVELQSGARYEATVKDIDHKLDLALIKIEPDTELPVLLLGRSSDLRAGEFVVALGSPFSLQNTVTAGIVSTTQRGGRELGLKNSDIDYIQTDAIINHGNSGGPLVNLDGDVIGINTLKVTAGISFAIPSDRIRQFLEDYHERQLKGKAPLQKKYLGLRMLPLTLNLLQEMKRQDPEFPDVSSGVFVYEVIQGSAAASSGLRDHDVIVSINGQPVTTTTDVIEAVKDNDFLSIIVLRGSQTLFLTVTPEIINYW505.95 HC FR2 (SEQ ID NO: 17) WVRQAPGKGLEWVGLight chain HVRs for Anti-HtrA1 Antibody YW505.94a.28 HVR L1:(SEQ ID NO: 18) RASQSINTYLA HVR L2: (SEQ ID NO: 2) SASFLYS HVR L3:(SEQ ID NO: 19) QQSDDTPPTHeavy chain HVRs for Anti-HtrA1 Antibody YW505.94a.28 HVR H1:(SEQ ID NO: 20) GFSISGYYIH HVR H2: (SEQ ID NO: 5) WIDPYGGDTNYADSVKGHVR H3: (SEQ ID NO: 6) GTFLTSWGHYFDYLight chain VR for Anti-HtrA1 Antibody YW505.94a.28 (SEQ ID NO: 28)DIQMTQSPSSLSASVGDRVTITCRASQSINTYLAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSDDTPPTFGQGTKVEIKRHeavy chain VR for Anti-HtrA1 Antibody YW505.94a.28 (SEQ ID NO: 29)EVQLVESGGGLVQPGGSLRLSCAASGFSISGYYIHWVRQAPGKGLEWVGWIDPYGGDTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGTFLTSWGHYFDYWGQGTLight chain HVRs for Anti-HtrA1 Antibody YW505.94a.54 HVR L1:(SEQ ID NO: 21) RASQVVGNYLA HVR L2: (SEQ ID NO: 2) SASFLYS HVR L3:(SEQ ID NO: 22) QQSDDHPPTHeavy chain HVRs for Anti-HtrA1 Antibody YW505.94a.54 HVR H1:(SEQ ID NO: 20) GFSISGYYIH HVR H2: (SEQ ID NO: 5) WIDPYGGDTNYADSVKGHVR H3: (SEQ ID NO: 6) GTFLTSWGHYFDYLight chain VR for Anti-HtrA1 Antibody YW505.94a.54 (SEQ ID NO: 30)DIQMTQSPSSLSASVGDRVTITCRASQVVGNYLAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSDDHPPTFGQGTKVEIKRHeavy chain VR for Anti-HtrA1 Antibody YW505.94a.54 (SEQ ID NO: 29)EVQLVESGGGLVQPGGSLRLSCAASGFSISGYYIHWVRQAPGKGLEWVGWIDPYGGDTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGTFLTSWGHYFDYWGQGTLight chain HVR Consensus Sequences HVR L1: (SEQ ID NO: 23)RASQX_(a)X_(b)X_(c)X_(d)X_(e)X_(f)A, whereinX_(a) is D, S or V; X_(b) is V or I; X_(c) is S, N or G; X_(d) is T or N; X_(e) is A or Y; and X_(f) is V or L;HVR L2: (SEQ ID NO: 2) SASFLYS HVR L3: (SEQ ID NO: 24)QQX_(g)X_(h)X_(i)X_(j)PX_(k)T, whereinX_(g) is S, V or D; X_(h) is Y, D or S; X_(i) is T, S, A, D or N; X_(j) is T, H, N, S, A, L or R; andX_(k) is P, T, A or S; Heavy chain HVR Consensus Sequences HVR H1:(SEQ ID NO: 25) GFX_(l)IX_(m)X_(n)YYTIH, whereinX_(l) is N, S or T; X_(m) is S, D, Y or A; and X_(n) is G or D; HVR H2:(SEQ ID NO: 26) WIDPYGGDTX_(o)YADSVKG, wherein X_(o) is N or D; HVR H3:(SEQ ID NO: 27) GTFLTX_(p)WGHYFDY, wherein X_(p) is S or T.Consensus Light chain VR (SEQ ID NO: 31)DIQMTQSPSSLSASVGDRVTITCRASQX_(a)X_(b)X_(c)X_(d)X_(e)X_(f)AWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQX_(g)X_(h)X_(i)X_(j)PX_(k)TFGQGTKVEIKR,whereinX_(a) is D, S or V; X_(b) is V or I; X_(c) is S, N or G; X_(d) is T or N; X_(e) is A or Y; X_(f) iS V or L;X_(g) is S, V or D; X_(h) is Y, D or S; X_(i) is T, S, A, D or N; X_(j) is T, H, N, S, A, L or R;and X_(k) is P, T, A or S; Consensus Heavy chain VR (SEQ ID NO: 32)EVQLVESGGGLVQPGGSLRLSCAASGFX_(l)IX_(m)X_(n)YYIHWVRQAPGKGLEWVGWIDPYGGDTX_(o)YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGTFLTX_(p)WGHYFDYWGQ GT,whereinX_(l) is N, S or T; X_(m) is S, D, Y or A; X_(n) is G or D; X_(o) is N or D;and X_(p) is S or T YW505.94a51 HVR L1: (SEQ ID NO: 33) RASQDVGTYLAYW505.94a.1 HVR L3: (SEQ ID NO: 34) QQVYSHPPT YW505.94a.7 HVR L3:(SEQ ID NO: 35) QQSYTNPPT YW505.94a.22 HVR L3: (SEQ ID NO: 36) QQSYATPTTYW505.94a.37 HVR L3: (SEQ ID NO: 37) QQSYSSPAT YW505.94a.39 HVR L3:(SEQ ID NO: 38) QQVYTTPPT YW505.94a.40 HVR L3: (SEQ ID NO: 39) QQVYATPSTYW505.94a.42 HVR L3: (SEQ ID NO: 40) QQSYNSPAT YW505.94a.46 HVR. L3:(SEQ ID NO: 41) QQSYSTPAT YW505.94a.50 HVR L3: (SEQ ID NO: 42) QQSYTAPTTYW505.94a.51 HVR L3: (SEQ ID NO: 43) QQDSTLPPT YW505.94a.52 HVR L3:(SEQ ID NO: 44) QQSDAAPPT YW505.94a.78 HVR L3: (SEQ ID NO: 45) QQSYSTPPTYW505.94a.89 HVR L3: (SEQ ID NO: 46) QQSYTRPPT YW505.94a.7 HVR H1:(SEQ ID NO: 47) GFSISDYYIH YW505.94a.26 HVR H1: (SEQ ID NO: 48)GFSIDGYYIH YW505.94a.38 HVR H1: (SEQ ID NO: 49) GFTIYDYYIHYW505.94a.78 HVR H1: (SEQ ID NO: 50) GFSIAGYYIH YW505.94a.82 HVR H1:(SEQ ID NO: 51) GFTISDYYIH YW505.94A.42 HVR H2: (SEQ ID NO: 52)WIDPYGGDTDYADSVKG YW505.94A.46 HVR H3: (SEQ ID NO: 53) GTFLTTWGHYFDY

TABLE A HVR and variable domain sequences for anti-HtrA1 YW505.94antibody and affinity-improved variants thereof. SEQ ID NOs H1 H2 H3 L1L2 L3 HC VR LC YR Consensus 25 26 27 23 2 24 32 31 YW505.94 4 5 6 1 2 38 7 YW505.94a 20 5 6 1 2 3 29 7 YW505.94a.28 20 5 6 18 2 19 29 28YW505.94a.54 20 5 6 21 2 22 29 30 YW505.94a.1 20 5 6 1 2 34 YW505.94a.747 5 6 1 2 35 YW505.94a.22 20 5 6 1 2 36 YW505.94a.26 48 5 6 1 2 3YW505.94a.37 47 5 6 1 2 37 YW505.94a.38 49 5 6 1 2 3 YW505.94a.39 20 5 61 2 38 YW505.94a.40 20 5 6 1 2 39 YW505.94a.42 20 52 6 1 2 40YW505.94a.46 20 5 53 1 2 41 YW505.94a.47 20 52 6 1 2 3 YW505.94a.50 47 56 1 2 42 YW505.94a.51 20 5 6 33 2 43 YW505.94a.52 20 5 6 1 2 44YW505.94a.77 20 5 53 1 2 3 YW505.94a.78 50 5 6 1 2 45 YW505.94a.82 51 56 1 2 3 YW505.94a.89 20 52 6 1 2 46

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1: Generation of Anti-HtrA1 Antibodies

Phage Display.

Synthetic antibody libraries displayed bivalent Fab fragments on the M13phage and the diversity was generated by use of oligo-directedmutagenesis in three complementarity determining regions (CDRs) of theheavy chain. The details of the Fab libraries were described previously(Lee, C. V., et al., J Mol Biol. 340:1073-93 (2004); Lee, C. V., et al.,J Immunol Methods. 284:119-32 (2004)). Nunc 96-well Maxisorpimmunoplates were coated overnight at 4° C. with MuHtrA1_PD (10 μg/ml)and then blocked for 1 h at room temperature with phage blocking buffer(PBS, 1% (w/v) BSA, 0.05% (v/v) Tween 20). The antibody phage librarieswere added to the MuHtrA1_PD-coated plates and incubated overnight atroom temperature. The plates were washed with PBS, 0.05% (v/v) Tween-20buffer and bound phage were eluted with 50 mM HCl-500 mM NaCl for 30 minand neutralized with an equal volume of 1 M Tris-HCl, pH 7.5. Recoveredphage was amplified in E. coli XL-1 blue cells. During subsequentselection rounds, incubation of antibody phage with the antigen-coatedplates was reduced to 2-3 hours and the stringency of plate washing wasgradually increased. 13 identified clones were reformatted to IgG (asdescribed below for Fab94), expressed in 293 cells and purified byProtein A affinity chromatography. The 13 purified IgG bound toMuHtrA1_PD in a binding ELISA.

Anti-HtrA1 Fab94 and IgG94 Expression and Purification.

The variable regions of both the heavy and light chains of Fab94(YW505.94) were subcloned into a E. coli Fab expression vector (AEP1).The resulting plasmid was transformed into E. coli. strain 34B8. Thesingle colony was grown overnight at 37° C. in 30 ml LB mediumsupplemented with 50 μg/ml of Carbenicilin. Five ml of the culture wasinoculated into 500 ml of complete C.R.A.P. medium (Simmons, L. C., etal., J Immunol Methods. 263:133-47 (2002)) supplemented withCarbenicilin (50 μg/ml) and grown at 30° C. for 24 h. The Fab94 proteinwas purified using protein A agarose resin.

The variable domains of both the light chain and heavy chain of Fab94were cloned into a pRK5-based plasmid with human light chain or heavychain (human IgG1) constant domain for transient expression in Chinesehamster ovary (CHO) cells. The IgG94 protein was purified by use ofprotein A agarose chromatography.

The amino acid sequences for the light chain variable region and heavychain variable region for Fab94 (YW505.94) are shown in FIGS. 1A and 1B,respectively.

Example 2: Production of HtrA1 Proteins

Protein Constructs.

Full length human HtrA1 (HuHtrA1) (Q23-P480) and full length murineHtrA1 (MuHtrA1) (P24-P480) were cloned by PCR amplification from fulllength clones using Taq polymerase. The primers contained thenucleotides required for adding portions of the restriction sites BamHIand EcoRI to the resulting PCR products. The PCR fragments were ligatedinto a modified pAcGP67 baculovirus transfer vector (BD Pharmingen)containing a His₆ sequence (SEQ ID NO: 90) upstream of the BamHIrestriction site resulting in an N-terminal His₆ tag (SEQ ID NO: 90) onthe HtrA1 proteins.

The protease domain of human HtrA1 (HtrA1_PD) lacking the N-domain andthe PDZ domain (e.g., containing residues D161-K379) was cloned by PCRamplification. The amplified DNA was inserted into a modified pET vectorresulting in an N-terminal His₆ tag (SEQ ID NO: 90) and a thrombincleavage site fused upstream of the D161 codon of HtrA1. HtrA1_PD Alamutants were generated using the QuikChange XL site-directed mutagenesiskit according to the manufacturer's protocol (Agilent Technologies,Santa Clara Calif.). Constructs were verified by DNA sequencing. Theprotease domains of murine HtrA1 (MuHtrA1_PD) (G156-S367), murine HtrA3(MuHtrA3_PD) (P133-F350) and murine HtrA4 (MuHtrA4_PD) (E159-Y371) werecloned from full length clones using Pfu (Agilent) polymerase. Theprimers contained the nucleotides required for adding the restrictionsites Not I and Stu I to the resulting PCR products. The PCR fragmentswere ligated into pST23 of an expression vector with a phoA promoter.This expression vector contains the amino acid sequences MK(HQ)₆MHQSTAA(SEQ ID NO: 11) upstream of the Not I restriction site resulting inN-terminal fusion of unizyme tag to the protease domains.

As described herein, amino acid residues of huHtrA1, muHtrA1, muHtrA3,and muHtrA4 are made with reference to SEQ ID NOs:13-16, respectively.Amino acids positions are specified by the one letter amino acid codefollowed by its position within one of SEQ ID NOs: 13-16. For example,full length huHtrA1 comprises a sequence starting at glutamine atposition 23 of SEQ ID NO:13 and ending at proline at position 480 of SEQID NO:13, e.g., Q23-P480. Similarly, mutations at a particular positionwithin an HtrA1 protein are designated by the starting amino acid,followed by the position within one of SEQ ID NOs:13-16, followed by thesubstituted amino acid. For Example, a mutation of Lysine at position248 of HuHtrA1 (SEQ ID NO:13) to alanine is referred to as K248A.

Protein Expression and Purification.

HuHtrA1 and MuHtrA1 were expressed in Trichoplusia ni insect cells(Expression Systems LLC, Woodland, Calif.). Harvested insect cell media(after pH was adjusted to 6.8) had NiCl₂, CaCl₂, and NaCl added to finalconcentrations of 1.0, 2.5 and 150 mM respectively.

HuHtrA1_PD constructs (wildtype and S328A mutant) were expressed in E.coli BL21 (Stratagene). E. coli cultures were grown at 37° C. in LBmedium containing 50 μg/ml carbenicillin until A₆₀₀ reached 0.8 to 1.0and then induced with 0.4 mM IPTG and grown at 16° C. for 24 h. Thebacterial cell pellets were resuspended in 1/10^(th) culture volume of50 mM Tris pH 8.0, 500 mM NaCl and disrupted using a Microfluidizer.Lysates were centrifuged at 30,000×g for 30 min.

HuHtrA1, MuHtrA1, HuHtrA1_PD and HuHtrA1_PD(S328A) were purified usingnickel-nitrilo-triacetic acid resin (Qiagen). After loading insect cellmedia or E. coli lysates, columns were washed with 10 column volumes(CV) of 50 mM Tris pH 8.0, 500 mM NaCl followed by 10 CV of 50 mM TrispH 8.0, 1 M NaCl, 20 mM imidazole. Proteins were eluted with 50 mM TrispH 8.0, 200 mM NaCl, 10% glycerol, 0.25% CHAPS, 300 mM imidazole. Pooledfractions were further purified by size exclusion chromatography on aS-200 column (GE Healthcare) and peak fractions corresponding to thetrimeric protein forms were collected. Protein purity was greater than90% as assessed by SDS-PAGE and protein concentrations were determinedusing the BCA™ protein assay (Pierce, Rockford, Ill.).

HuHtrA1_PD Ala mutants (panel of 15 mutants except S328A) were expressedas described for the wildtype form of HuHtrA1_PD (see above). Lysisbuffer (PopCulture, EMD Chemical, San Diego Calif.) was added tobacterial cell pellets at 50 ml lysis buffer/500 ml pellet. The pelletwas suspended completely in lysis buffer with a polytron and stirred atroom temperature for 30 min. The cell lysate was centrifuged (Beckman,LA-16.250 rotor) at 33,700×g for 30 min at 4° C. and the resultingsupernatant was filtered through a 0.22 μm filter unit (Nalgene). Thefiltered supernatant was loaded onto a Ni-NTA column (3 ml, Qiagen)pre-equilibrated with 50 mM Tris, 300 mM NaCl, 10 mM imidazole, 10%glycerol pH 8.0 (buffer A). Once loaded the column was washed with 12 CVof buffer A followed by 20 CV of buffer A+0.1% Triton X-114. The columnwas washed again with 15 CV of buffer A to remove detergent. Proteinswere eluted with buffer A+200 mM imidazole, 0.25% CHAPS. PooledFractions were further purified with Superdex 200 (Hi load 16/60, 120ml, GE Healthcare) equilibrated with 50 mM Tris pH 8.0, 200 mM NaCl, 10%glycerol, 0.25% CHAPS. The fractions corresponding to the HtrA1_PDtrimer peak were pooled. Protein purity was greater than 90% as assessedby SDS-PAGE and protein concentrations were determined by use of theBCA™ protein assay (Pierce, Rockford, Ill.).

MuHtrA1_PD, MuHtrA3_PD and MuHtrA4_PD were expressed in E. coli 58F3(Szeto, W., et al., Cancer Res. 61:4197-205 (2001)). Overnight E. colicultures, grown at 30° C. in LB medium containing 50 μg/mlcarbenicillin, were diluted (1/100 vol) into a larger culture containingC.R.A.P. medium (Simmons, L. C., et al., J Immunol Methods. 263:133-47(2002)) supplemented with 50 μg/ml carbenicillin. After inoculation E.coli were grown for approximately 20 h at 30° C. MuHtrA1_PD was purifiedas described for HuHtrA1_PD (see above). For purification of MuHtrA3_PDand MuHtrA4_PD, lysis buffer (50 mM Tris pH 8.5, 500 mM NaCl, 10 mMimidazole, 10% glycerol) was added to the bacterial cell pellet. Thepellet was homogenized in lysis buffer with a polytron and cellsdisrupted with a mircofludizer. The cell lysate was centrifuged and theresulting supernatant filtered through a 0.22 μm filter unit (Nalgene).The filtered supernatant was loaded onto a 3 ml Ni-NTA column (Qiagen)pre-equilibrated with 25 mM Tris pH 8.5, 500 mM NaCl, 20 mM imidazole,10% glycerol (buffer B). After loading, the column was washed with 12 CVof buffer B followed by 20 CV of buffer B+0.1% Triton X-114. The columnwas washed again with 15 CV of buffer B to remove detergent. Proteinswere eluted with 25 mM Tris pH 8.5, 500 mM NaCl, 250 mM imidazole, 10%glycerol, 0.25% CHAPS. Pooled fractions were further purified on a 120ml Superdex 75 (GE Healthcare) equilibrated with 25 mM Tris pH 8.5, 10%glycerol, 0.5 mM TCEP, 0.25% CHAPS. The fractions corresponding to theprotease trimer peak were pooled. Protein purity was greater than 90% asassessed by SDS-PAGE.

Example 3: Determination of Inhibitory Activity of Anti-HtrA1 AntibodiesUsing a FRET Assay

Synthesis of Peptide Substrate.

The peptide H2-Opt (Mca-IRRVSYSF(Dnp)KK) (SEQ ID NO:12), originallydescribed as a substrate for HtrA2 (Martins, L. M., et al., J Biol Chem.278:49417-27 (2003)), was synthesized on Fmoc-Lys(Boc)-wang resin usingstandard coupling procedures with HBTU. Fmoc-Lys(DNP)-OH (Anaspec) wasincorporated in the P5′ position. The peptide was synthesized up to P5(Mca, 7-Methoxy-coumarin, Aldrich) and then cleaved from the solidsupport using trifluoroacetic acid, triisopropylsilane and water for 2hours at room temperature. Peptide was precipitated from ethyl ether,extracted with acetic acid, acetonitrile, water and lyophilized. Crudelabeled peptide was dissolved and purified on preparative reverse phaseC18 column using acetonitrile/water. Purified fractions were pooled,lyophilized and analyzed by liquid chromatography/mass spectrometry(PE/Sciex) and found to be consistent with their calculated masses.

Enzymatic Assays with Peptide Substrate.

HtrA1 was incubated in 96-well black optical bottom plates (Nalge NuncInt., Rochester, N.Y.) with IgG94 or Fab94 serially diluted in 50 mMTris-HCl, pH 8.0, 200 mM NaCl, 0.25% CHAPS (assay buffer) for 20 min at37° C. For initial testing a panel of 13 phage derived anti-HtrA1antibodies, single concentrations of IgGs (final concentrations 0.16mg/ml-0.28 mg/ml) were incubated with HuHtrA1 or HuHtrA1_PD. A 10 mMstock solution of the peptide substrate Mca-IRRVSYSF(Dnp)KK (SEQ IDNO:12) (H2-Opt) in DMSO was diluted in water to 12.5 μM, pre-warmed at37° C. and then added to the reaction mixture. The final concentrationsof the reactants were: 5 nM HuHtrA1 or MuHtrA1, 0.005-300 nM IgG94,0.02-900 nM Fab94, 2.5 μM H2-Opt. The increase of fluorescence signal(excitation 328 nm, emission 393 nm) was measured on a SPECTRAmax M5microplate reader (Molecular Devices, Sunnyvale, Calif.) and the linearrates of H2-Opt cleavage (mRFU/min) determined. Experiments with IgG94inhibition of trypsin (Roche) and elastase (MP Biomedicals) were carriedout identically, except that the final enzyme concentrations were 1 nM,the IgG94 concentration was 300 nM and the incubation time was 15 min.

As shown in FIG. 2, a panel of 13 phage derived antibodies (IgG) wasincubated with HuHtrA1 or HuHtrA1_PD and enzyme activity measured. Ofthe 13 antibodies tested, antibodies YW503.57, YW504.57, YW504.61 andYW505.94 (also referred to as Fab94, antibody 94, Ab94 or IgG94)strongly inhibited both HuHtrA1 and HuHtrA1_PD activities.

As shown in FIG. 3, IgG94 inhibited the enzymatic activity of HuHtrA1towards the H2-Opt substrate in a concentration-dependent fashion withan IC₅₀ value of 1.8 nM. Complete inhibition was achieved above 30 nMIgG94. In contrast, two other members of the trypsin-like serineprotease family, trypsin and elastase, were not inhibited by IgG94 at300 nM, indicating that IgG94 has specificity towards HuHtrA1. Fab94also inhibited HuHtrA1 in a concentration-dependent manner, but lesspotently than IgG94 as indicated by the IC₅₀ value of 29.1 nM. Theseresults are in excellent agreement with the K_(D) value of 31.1 nMdetermined by surface plasmon resonance experiments (see Table 1 below).The results show that IgG94 and Fab94 are able to completely neutralizethe enzymatic activity of HuHtrA1 towards a small synthetic peptidesubstrate and that this activity is specific. Moreover, the about16-fold increase in potency of IgG94 vs. Fab94 is consistent with theavidity effects predicted from the ‘cage-like’ IgG94:HtrA1 complex basedon mass analysis (see e.g., Table 2 and FIG. 9).

The inhibitory potencies of affinity-improved variant antibodyYW505.94a.28 was also determined using the assay described above withthe fluorescence quenched peptide substrate H2-Opt and 1.0 nM of HuHtrA1or 1.5 nM of HuHtrA1_PD. The results are shown below.

HuHtrA1_PD HuHtrA1 YW505.94a.28 format IC₅₀ nM IC₅₀ nM IgG 0.229 0.335Fab 2.03 1.95

Example 4: Determination of Inhibitory Activity of Anti-HtrA1 AntibodiesUsing Macromolecular Substrates

Bovine β-casein (Sigma-Aldrich) was repurified on a MonoQ ion exchangecolumn to yield highly purified material. HuHtrA1 was incubated inEppendorf tubes together with increasing concentrations of IgG94 inassay buffer for 15 min at 37° C. after which the macromolecularsubstrates were added. For β-casein digestion, the final concentrationof reactants were: 10 nM HuHtrA1, 50 μg/ml β-casein, 2.3-150 nM IgG94.For decorin the final concentrations were: 125 nM HuHtrA1, 50 μg/mldecorin (R & D Systems), 2-125 nM IgG94. For biglycan the finalconcentrations were: 75 nM HuHtrA1, 50 μg/ml biglycan (R & D Systems),2.3-150 nM IgG94. After incubation at 37° C. (2 h for β-casein, 14 h fordecorin, 6 h for biglycan), SDS sample buffer was added and samplesboiled and analyzed by SDS-PAGE (non-reducing).

Hydrolysis of fluorescent dye-labeled casein, BODIPY FL casein(Invitrogen), was carried out in 96-well black optical bottom plates(Nalge Nunc Int., Rochester, N.Y.). HuHtrA1 or MuHtrA1 were incubatedwith IgG94 serially diluted in assay buffer for 15 min at 37° C. Afteraddition of BODIPY FL in assay buffer, the reactant concentrations wereas follows: 30 nM HuHtrA1, 30 nM MuHtrA1, 5 μg/ml BODIPY FL, 0.24-250 nMIgG94. The increase of fluorescence signal (excitation 484 nm, emission535 nm) was measured on a SPECTRAmax M5 microplate reader (MolecularDevices, Sunnyvale, Calif.) and the linear rates of BODIPY FL cleavage(mRFU/min) determined and expressed as percentage of uninhibited rates(control).

As shown in FIG. 6, HuHtrA1 degraded the macromolecular substratesβ-casein, decorin and biglycan (lane 2). In the absence of HuHtrA1, thesubstrates remained intact during the experimental period (FIG. 6, lane1). Preincubation of HuHtrA1 with increasing concentrations of IgG94(FIG. 6, lane 9-lane 3) resulted in a concentration-dependent inhibitionof substrate degradation. At the highest IgG94 concentrations tested,degradation of β-casein, decorin and biglycan was completely prevented(FIG. 6, lane 3). The results show that IgG94 potently and effectivelyinhibited HuHtrA1 activity towards three macromolecular substrates.

As shown in FIG. 4, IgG94 concentration-dependently inhibited BODIPY FLhydrolysis by both HuHtrA1 and MuHtrA1 with IC₅₀ values of 8.3 nM and5.4 nM, respectively. The results show that IgG94 recognizes andcompletely neutralizes the enzymatic activities of both HuHtrA1 andMuHtrA1 towards the macromolecular substrate BODIPY FL.

Example 5: Determination of the Specificity of Anti-HtrA1 Antibodies toHtrA1

The specificity of IgG94 was assessed by measuring its activity forinhibiting muHtrA1_PD as compared to its ability to inhibit thestructurally related proteases MuHtrA3_PD and MuHtrA4_PD (see FIG. 5).The specificity was determined using the FRET assay as described abovein Example 3, except that the concentrations of MuHtrA1_PD, MuHtrA3_PDand MuHtrA4_PD 6 nM, 20 nM and 20 nM, respectively, and theconcentration range of IgG94 was 0.2-400 nM. The specificity was alsoanalyzed using the BODIPY FL casein substrate as described above inExample 4, except that the concentration of MuHtrA1_PD, MuHtrA3_PD andMuHtrA4_PD was 50 nM and the concentration range of IgG94 was 1.4-1000nM. IgG94 inhibited MuHtrA1_PD cleavage of H2-Opt (FIG. 5, top panel)and BODIPY FL (FIG. 5, bottom panel), but did not inhibit cleavage ofthe same substrates by MuHtrA3_PD and MuHtrA4_PD up to concentrations of400 nM and 1000 nM for H2-Opt and BODIPY FL substrates, respectively.The results suggest that IgG94 has excellent specificity and does notimpair activities of related proteases.

Example 6: Determination of Antibody Affinity by BIAcore

To determine the binding affinity of Fab94 by single-cycle kinetics,Surface Plasmon Resonance (SRP) measurement with a BIAcore™ TI100instrument was used. Briefly, series S sensor chip CM5 was activatedwith EDC and NHS reagents according to the supplier's instructions, andstreptavidin (Pierce) was coupled to achieve approximately 2000 responseunits (RU), followed by blocking unreacted groups with 1M ethanolamine.

For kinetics measurements, biotinylated HuHtrA1_PD or MuHtrA1_PD werefirst injected at 10 μl/min flow rate to capture approximately 100 RU at3 different flow cells (FC), except for FC1 (reference cell), and then5-fold serial dilutions of Fab94 (0.48 nM-300 nM) in 0.01M HEPES pH 7.4,0.15M NaCl, 0.005% surfactant P20 were injected (flow rate: 30 dl/min)with no regeneration between injections. The sensorgrams were recordedand evaluated by BIAcore™ T100 Evaluation Software (version 2.0) aftersubtraction of reference cell signal. Association rates (k_(on)) anddissociation rates (k_(off)) were calculated using a simple one-to-oneLangmuir binding model. The equilibrium dissociation constant (K_(D))was calculated as the ratio k_(off)k_(on) (see Table 1 below). Thebinding kinetics of Fab94 to HuHtrA1_PD and to MuHtrA1_PD were verysimilar. The K_(D) values were 31.1 nM and 28.9 nM, respectively.

TABLE 1 Binding Affinities of Fab94 to HuHtrA1_PD and MuHtrA1_PD bySurface Plasmon Resonance. k_(on) k_(off) K_(D) (10⁵ M⁻¹s⁻¹) (10⁴ s⁻¹)(nM) HuHtrA1 PD 3.5 109.0 31.1 MuHtrA1 PD 4.5 129.0 28.9

Example 7: Epitope Mapping of IgG94 on HuHtrA1_PD

To identify the functional binding epitope of IgG94, a panel ofHuHtrA1_PD mutants with individual alanine mutations was generated forbinding experiments. Most of the mutated residues are located on surfaceloops surrounding the active site and also included the catalytic serine(S328) and aspartate (D250). The mutants were expressed in E. coli andthe trimers purified by size exclusion chromatography as described inExample 2. Binding was determined using an ELISA assay, which wasconducted using a MAXISORP™ microtiter plate coated with HuHtrA1_PDmutants at 2 μg/ml in PBS for 1 h, followed by blocking with PBST buffer(0.5% BSA and 0.05% Tween 20 in PBS) for 1 h at room temperature.Five-fold serially diluted Ig94 (50 nM to 0.0006 nM) in PBST buffer wasadded and incubated for 30 min. The plates were washed with PBT buffer(0.05% Tween 20 in PBS), and HRP-conjugated goat anti-human IgG (H+L)(Invitrogen) was added (1:5000 in PBST buffer) and incubated for 1 h.The plates were washed with PBT buffer and developed by addingtetramethylbenzidine substrate (Kirkegaard and Perry Laboratories,Gaithersburg, Md.). The absorbance at 450 nm was plotted as a functionof antibody concentration in solution to determine EC₅₀ values. Thelocation of mutants effecting binding were then mapped onto thestructure of HtrA1.

In the binding ELISA, IgG94 showed the greatest reduction in binding tothe mutants HuHtrA1_PD(N224A) and HuHtrA1_PD(K248A) (see FIG. 7A). Uponrepetition of the experiment using a lower concentration of HtrA1 tocoat the plates (1 μg/ml) and a shortened incubation time (reduced from1 hour to 20 minutes incubation), IgG94 also showed at least a 5-foldreduction in binding to HuHtrA1 mutants V201A, H220A, T223A, K225A,K243A and E247A (see FIG. 7A). The results indicate that IgG94 binds toan epitope that includes residues N224 and K248, which are located onLoop B and Loop C (see FIGS. 7B and 7C). In trypsin-like serineproteases these loops are known to be part of the specificitydetermining region that interacts with substrates. K248 (Loop C) is veryclose to the catalytic D250 and N224 to the catalytic H220. Therefore,binding of IgG94 to these residues may impair substrate access to thecatalytic cleft either by direct steric hindrance or by an indirectallosteric mechanism as found for neutralizing anti-HGFA antibodies thatalso recognize residues in Loop C (Ganesan, R., et al., Structure17:1614-1624 (2009); Wu, Y., et al., Proc Natl Acad Sci USA 104:19784-9(2007)). Alternatively, antibody binding may inhibit catalysis bydirectly influencing the catalytic triad residues D250 and/or H220.

Example 8: Stoichiometry of Complexes of HtrA1_PD with Fab94 and IgG94by Size Exclusion Chromatography-Multi-Angle Light Scattering(SEC-MALLS)

A catalytically inactive form HuHtrA1_PD(S328A) was used for complexformation. The Fab94:HuHtrA1_PD(S328A) and IgG94:HuHtrA1_PD(S328A)complexes were formed in Tris buffer (50 mM Tris-HCl, pH 8.0, 200 mMNaCl, 10% glycerol, 0.25% CHAPS). The complexes and the individualcomponents alone were incubated at 4° C. overnight and then injected andresolved on a S200 Superdex 10/300 GL column (GE Healthcare) in Trisbuffer with a flow rate of 0.5 mL/min. Data was collected with an18-angle light scatter detector (Dawn Helios II with QELS) and arefractive index detector (Optilab reX) from Wyatt Technologies.Analysis was done with Astra 5 software to yield molar massindependently ofelution time. Normalization was performed with a controlIgG.

SEC-MALLS experiments show that Fab94 and IgG94 form complexes withHuHtrA1_PD(S328A) that have distinct masses. The determined masses anddeduced stoichiometry of the complexes are shown in Table 2.

TABLE 2 Stoichiometry of HuHtrA1_PD (S328A) in complex with Fab94 andIgG94 determined by SEC-MALLS. Mass by Expected Mass MALLS Best-fit massdifference Components* (Da)** stoichiometry^(&) (Da) (%) HtrA1_PD 76,755— — — Fab94 47,400 — — — IgG94 151,200 — — — Fab94:HtrA1_PD 210,100 3:1218,955 4.2 IgG94:HtrA1_PD 618,600 3:2 607,110 1.9 *HuHtrA1_PD wasHuHtrA1_PD with the catalytic serine mutated to alanine(HuHtrA1_PD(S328A)); the mass represents the trimer **Average of twoindependent experiments ^(&)Ratio of antibody:protease trimer

The determined mass of the Fab94:HtrA1_PD(S328A) complex of 210,100 Dais consistent with a complex of 3 Fabs binding to one HuHtrA1_PD(S328A)trimer (3:1 complex). The difference between the experimental and thetheoretical masses of such a 3:1 complex is only 4.2%. Therefore, oneFab is able to bind to each HuHtrA1_PD(S328A) monomer within one HtrA1homo-trimer.

The determined mass of the IgG94:HtrA1_PD(S328A) complex of 618,600 Dafits well to a 3:2 stoichiometry, in that 3 IgG molecules bind to twoHuHtrA1_PD(S328A) trimers. The difference between the experimental andthe theoretical mass of such a 3:2 complex is only 1.9%.

The elucidated stoichiometries are in agreement with the findings thatFab94 and IgG94 are able to completely inhibit HtrA1 activity, in thateach monomer within an HtrA1 trimer is binding to one Fab or to one Fabarm of an IgG. Thus, in these complexes there is no ‘free’ HtrA1 monomeravailable, in agreement with the complete inhibition of HtrA1 enzymeactivity by Fab94 and IgG94. We propose a ‘cage’ model for theIgG94:HtrA1_PD(S328A) complex in which the Fab arms of the 3 IgGs arebridging two trimers, each Fab arm binding to one monomer (see FIG. 9).The model also accounts for the about 16-fold increased potency of IgG94over Fab94 in enzymatic assays (see e.g., FIG. 3) in that IgG94,strongly benefits from avidity effects in its binding to HtrA1_PDtrimers.

Example 9: Affinity Maturation of YW505.94

Construct Libraries for Anti-HtrA1 Affinity Maturation.

Clone YW505.94a was derived from YW505.94 [Ab94] by changing Kabatresidue N28 to serine within CDR H1 in order to remove a potentialN-linked glycosylation site. Phagemid pW0703, derived from phagemidpV0350-2b (Lee et al., J. Mol. Biol 340, 1073-1093 (2004)), whichcontains a stop codon (TAA) in all CDR-L3 positions and displaysmonovalent Fab on the surface of M13 bacteriophage, served as a librarytemplate for grafting the heavy chain variable domain (V_(H)) of cloneYW505.94a for affinity maturation. Both hard and soft randomizationstrategies were used for affinity maturation. For hard randomization,one light chain library (L1/L2/L3hard) with selected positions at threelight chain CDRs was randomized using amino acids designed to mimic anatural human antibody and the designed DNA degeneracy was as described(Lee et al., 2004). For soft randomization, selected residues at Kabatpositions 91, 92, 93, 94 and 96 of CDR-L3, 28-35 of CDR-H1, 50-58 ofCDR-H2, and 95-100 of CDR-H3 with two different combinations of CDRloops, L3/H1soft, L3/H2soft and L3/H3soft, were targeted forrandomization. To achieve the soft randomization conditions, whichintroduce a mutation rate of approximately 50% at the selectedpositions, the mutagenic DNA was synthesized with 70-10-10-10 mixturesof bases favoring the wild type nucleotides (Gallop et al., J. of Med.Chem. 37, 1233-1251 (1994)).

Phage Sorting Strategy to Isolate Affinity-Improved Variants.

For affinity improvement selection, phage libraries were subjected toplate sorting for the first round, followed by four rounds of solutionsorting. For the first round of plate sorting, four libraries(L1/L2/L3hard, L3/H soft, L3/H2soft and L3/H3soft) were sorted against ahuman HtrA1 (HuHtrA1) coated plate (NUNC Maxisorp plate) separately withphage input at about 3 O.D./ml in 1% BSA and 0.05% Tween 20 for 1 hourat room temperature (RT). After the first round of plate sorting, fourrounds of solution sorting were performed to increase the stringency ofselection. For solution sorting, 1 O.D./ml of phage propagated fromfirst round of plate sorting were incubated with 500 nM of biotinylatedHuHtrA1 in 100 ul buffer containing 1% Superblock (Pierce biotechnology)and 0.05% Tween20 for at least 1 hour at room termperature (RT). Themixture was further diluted 10× with 1% Superblock and applied 100ul/well to neutravidin-coated wells (10 μg/ml) for 15 min at RT withgentle shaking so that biotinylated HuHtrA1 could bind phage. The wellswere washed with PBS-0.05% Tween20 ten times. To determine backgroundbinding, control wells containing phage without biotinylated HuHtrA1selection were captured on neutravidin-coated plates. Bound phage waseluted with 0.1N HCl for 20 min, neutralized by 1/10 volume of 1M TrispH 11, titered, and propagated for the next round. Next, three morerounds of solution sorting were carried out using two methods ofincreasing selection stringency simultaneously. The first method, whichis for on-rate selection, decreases biotinylated target proteinconcentration from 10 nM to 0.1 nM. The second method, which is foroff-rate selection, adds excess amounts of non-biotinylated HuHtrA1protein (100˜1000 fold more) to compete off weaker binders. Also, thephage input was decreased (0.1˜0.5 O.D/ml) to lower the background phagebinding.

High Throughput Affinity Screening ELISA (Single Spot Competition).

Colonies were picked from the fifth round screening and grown overnightat 37° C. in 350 ul/well of 2YT media with 50 μg/ml carbenicillin and 1e10/ml KO7 in 96-well block (QIAgene). From the same plate, a colony ofXL-1 infected parental phage was picked as a control. 96-well NuncMaxisorp plates were coated with 100 ul/well of HuHtrA1 protein (2ug/ml) in PBS at RT for 2 hours. The plates were blocked with 100 ul of0.5% BSA and 0.05% Tween in PBS (PBST buffer) for one hour.

The phage supernatant was diluted 1:5 in PBST buffer with or without 5nM HuHtrA1 in 100 ul total volume and incubated at least 1 hour at RT.Then, 75 ul of mixture were transferred to the HuHtrA1 coated plates.The plate was gently shaken for 15 min to allow the capture of unboundphage to the HuHtrA1 coated plate. The plate was washed five times with0.05% Tween20 in PBS (PBT buffer). The binding was quantified by addinghorse radish peroxidase (HRP)-conjugated anti-M13 antibody in ELISAbuffer (1:5000) and incubated for 30 min at RT. The plates were washedwith PBT buffer five times. Next, 100 ul/well of a 1:1 ratio of 3,3′,5,5′-tetramethylbenzidine (TMB) Peroxidase substrate and PeroxidaseSolution B (H₂O₂) (Kirkegaard-Perry Laboratories (Gaithersburg, Md.))was added to the well and incubated for 5 minutes at RT. The reactionwas stopped by adding 100 ul 1 M Phosphoric Acid (H₃PO₄) to each welland allowed to incubate for 5 minutes at RT. The OD of each well wasdetermined using a standard ELISA plate reader at 450 nm. The ODreduction (%) was calculated by the following equation:

OD_(450 nm) reduction (%)=[(OD_(450 nm) of wells withcompetitor)/(OD_(450 nm) of well with no competitor)]*100

In comparison to the OD_(450 nm) reduction (%) of the well of parentalphage (100%), clones that had the OD_(450 nm) reduction (%) lower than50% were picked for sequence analysis (see FIGS. 15 and 16). Uniqueclones were selected for phage preparation to determine binding affinity(phage IC₅₀) by phage competition ELISA (see FIGS. 17 and 18). Then, themost affinity-improved clones (YW505.94a.28 & YW505.94a.54) werereformatted into human IgG1 for antibody production and further BIAcorebinding kinetic analysis.

Phage Competition ELISA to Determine IC₅₀.

MAXISORP™ microtiter plates were coated with HuHtrA1 at 2 μg/ml in PBSfor 2 hr at RT and then blocked with PBST buffer for another hour at RT.Purified phage from culture supernatants were incubated with seriallydiluted HuHtrA1 or MuHtrA1 in PBST buffer in a tissue-culture microtiterplate for an hour, after which 80 μl of the mixture was transferred tothe HuHtrA1-coated wells for 15 minutes to capture unbound phage. Theplate was washed with PBT buffer, and HRP-conjugated anti-M13 (AmershamPharmacia Biotech) was added (1:5000 in PBST buffer) for one hour. Theplate was washed with PBT buffer and developed by addingtetramethylbenzidine substrate (Kirkegaard and Perry Laboratories,Gaithersburg, Md.). The absorbance at 450 nm was plotted as a functionof HuHtrA1 or MuHtrA1 concentration in solution to determine phage IC₅₀.This was used as an affinity estimate for the Fab clone displayed on thesurface of the phage. FIG. 17 shows results from a phage competitionassay demonstrating the binding of YW505.94a affinity-improved variantsagainst HuHtrA1. FIG. 18 shows results from a phage competition assaydemonstrating the binding of YW505.94a affinity-improved variantsagainst MuHtrA1.

Antibody Affinity Determinations by BIAcore.

To determine the binding affinity of HtrA1 antibodies by single cyclekinetics, Surface Plasmon Resonance (SRP) measurement with a BIAcore™T100 instrument was used. Briefly, a series S sensor chip CM5 wasactivated with EDC and NHS reagents according to the supplier'sinstructions, and streptavidin (Pierce) was coupled to achieveapproximately 2000 response units (RU), followed by blocking un-reactedgroups with 1M ethanolamine.

For kinetics measurements, biotinylated human or murine HtrA1 was firstinjected at 10 ul/min flow rate to capture approximately 100 RU at 3different flow cells (FC), except for FC1 (reference), and then 5-foldserial dilutions of anti-HtrA1 Fab in HBS-P buffer (0.01M HEPES pH 7.4,0.15M NaCl, 0.005% surfactant P20) from low (0.48 nM) to high (300 nM)[5 points] were injected (flow rate: 30 μl/min) one after the other inthe same cycle with no regeneration between injections. The sensorgramwas recorded and subject to reference and buffer subtraction beforeevaluation by BIAcore™ T100 Evaluation Software (version 2.0).Association rates (km) and dissociation rates (k_(off)) were calculatedusing a simple one-to-one Langmuir binding model. The equilibriumdissociation constant (Kd) was calculated as the ratio k_(off)/k_(on).The results are shown in Table 3 below.

TABLE 3 Binding Affinities of Affinity Matured Fabs as Compared to theParent Fab. HuHtrA1 MuHtrA1 k_(on) k_(off) K_(D) k_(on) k_(off) K_(D)Fab (M⁻¹s⁻¹) (s⁻¹) (nM) (M⁻¹s⁻¹) (s⁻¹) (nM) YW505.94 3.51e5  109e−4 31.14.47e5  129e−4 28.9 YW505.94a 8.13e5 4.48e−2 55 1.48e6 8.56e−2 58YW505.94a.28 1.69e6 5.74e−3 3.4 2.02e6  9.3e−3 4.6 YW505.94a.54 2.97e67.84e−3 2.6 2.82e6 4.55e−3 1.6

Example 10: Sparing of Photoreceptors, Outer Nuclear Layer (ONL), andFunctional Responses in the Absence of HtrA1 Following Constant LightExposure

Construction of HtrA1 Knockout Mouse.

To generate HtrA1(+/−) embryonic stem cells, linearized targeting vectorDNA was electroporated into ES cells of Balb/c background to introduceFlox sites in the region 5′ of exon 1 and 3′ of exon 1 and an introducedneomycin-resistance gene (Neo). ES clones resistant to neomycin wereselected, and exon 1 plus the neomycin cassette was excised byelectroporating the ES cells with a cre-recombinase-expressingexpression plasmid. Homologous recombination was confirmed by sequencingof the entire HtrA1 locus and upstream promoter region in the targetedES cell. Targeted clones were injected into C57BL/6 blastocysts togenerate male chimeric mice of germline transmission. Interbreeding of+/− female and +/− male mice was performed to generate −/− female or −/−male HtrA1 mice on a Balb/c background. Successful deletion of HtrA1 wasconfirmed by RT-PCR of ovaries from HtrA1 wt and ko mice.

Constant Light Exposure Model.

Male Balb/c Htra1 wt/wt or ko/ko mice, 8-13 weeks old, are in normalhousing (<100 lux in cage) until start of constant light exposure (CLE).To start CLE, mice are housed singly in normal cages covered with a wirerack only, without a filter top. Food pellets and Hydrogels are placedon the bottom of the cage, and not on the wire top, for nourishment soas to not impede light entering the cage. Ten cages are placed on aMetro rack outfitted with fluorescent lights and enclosed in whitepanels to deliver ˜1200 lux to mice for up to 14 days. Cages are rotatedcounterclockwise around the rack daily during CLE to ensure equal lightexposure. If multiple shelves are used, cages are also rotated betweenshelves to ensure equal light exposure.

Optical Coherence Tomography.

Optical coherence tomography (OCT) is performed 4-7 days before lightexposure to provide a retinal thickness baseline measurement. OCT isperformed using a Heidelberg Spectralis HRA+OCT camera. Animals areanesthetized with 70-80 mg/kg ketamine/15 mg/kg xylazine in 150-300 ulsterile saline, eyes are dilated with 1% tropicamide drops and retinathickness is measured by OCT. Artificial tears are used to keep eyesmoist to prevent cataracts. After OCT, animals are allowed to recoverfrom anesthesia and returned to their cages and light rack.

Electroretinogram.

Electroretinograms (ERG) ERGs are performed 7 days before light exposureand at 15 days post CLE. ERGs are performed and recorded using aDiagnosys LLC Espion 2 visual electrophysiology system and a Colordomedesktop Ganzfeld as a light source. Mice are dark adapted overnight in adark room to equilibrate photoreceptors. Once dark adapted, allsubsequent procedures are performed in the dark with only a red lightfor illumination. Animals are anesthetized with Ketamine, 75-80 mg/kg, &Xylazine, 7.5-15 mg/kg, IP in 200-300 ul PBS. Mouse body temperature ismaintained at 37° C. using a homeothermic plate connected to its controlunit. Pupils are dilated with 1% tropicamide. ERGs from both eyes willbe recorded simultaneously with Burian-Allen silver or platinum wireloop electrodes. Mice are placed on a platform, a reference electrode isinserted subcutaneously through the forehead, and a ground electrode isinserted subcutaneously at the base of the tail. Gonak hypermellosesolution is placed on the cornea to establish an electrical contactbetween the cornea and the electrode, and protect eyes from dryingduring the experiment. Electrodes are placed on the left and right eyesand mouse inserted into a ColorDome light stimulator. Eyes will bestimulated with white light (7 flash intensities 4e-5, 2e-5, 0.5, 2, 5,10, 20 cd·s/m²) and signals bandpass-filtered at 0.15-1000 Hz andsampled at 2 kHz.

Determination of HtrA mRNA Levels in the Mouse Retina and Ovary.

Whole eyes were enucleated, the retina removed and placed in RLT Buffer(Qiagen) and frozen at −80° C. until homoginization. Retina's werehomogenized and shredded by gentleMACS M tubes (Miltinyi Biotec).Similar to the retina, ovarys were isolated from female HtrA1 wt, hetand ko mice and homogenized as described for the retina. RNA wasisolated using Qiagen Plus RNeasy kit; cDNA was generated with HighCapacity cDNA kit (Applied Biosystem); Taqman qPCR was performed using20 ug cDNA, Taqman Gene Expression Master Mix (Applied Biosystem) andTaqman Gene Expression Assay primers (Applied Biosystem) to exon 1-2(retina and ovary), exon 3-4 (ovary) and exon 5-6 (ovary) and ran on anABI 7500 Real-time PCR System (Applied Biosystem). HtrA1 expression wasnormalized to 18s expression (primer from Applied Biosystem).

As shown in FIG. 11, HtrA1 mRNA expression increases in a mouse model ofconstant light exposure. However, in the same model, HtrA2 levels do notincrease in the retina. In addition, HtrA1 levels are significantlyhigher than HtrA2, HtrA3 and HtrA4 levels in the retina of a non-exposedmouse.

In addition, we found that mice lacking HtrA1 expression show a sparingof photoreceptors (FIG. 13), the outer nuclear layer (ONL) (FIG. 14),and functional responses (ERG) (FIG. 12) in a mouse model oflight-induced degeneration.

Example 11: Effects of an Anti-HtrA1 Antibody in a Rat Model of ConstantLight Exposure

Rats will be subjected to constant light exposure as described above formice. The efficacy of an anti-HtrA1 antibody to protect the rat eye fromdegeneration caused by the light exposure will be evaluated. Rat eyeswill be subject to intravitreal injections of an anti-HtrA1 antibody ata dose and frequency suitable to maintain an effective concentration ofthe anti-HtrA1 antibody in the eye during the course of the experiment.The rat eyes will be monitored following light exposure to determineretinal thickness by OCT and functionality by ERG, as described abovefor the mouse model.

Example 12: HtrA1 Expression is Abundant in Rat Vitreous and Accumulatesin Mouse Eye Fluid and Eye Tissue Upon Light Stress

ELISA Assay.

For analysis of rat tissues, an anti-muHtrA1 rabbit polyclonal antibody(Genentech) was diluted to 125 ng/mL, while for analysis of mouse eyefluid and retina tissue, an anti-huHtrA1 mouse monoclonal antibody(Genentech) was diluted to 250 ng/mL, both in PBS, and coated onto384-well ELISA plates (Nunc; Neptune, N.J.) during an overnightincubation at 4° C. Plates were washed with PBS plus 0.05% Tween-20 andblocked during a two hour incubation with PBS plus 0.5% bovine serumalbumin (BSA). This and all subsequent incubations were performed atroom temperature with gentle agitation. Recombinant murine HtrA1standard (Genentech) and samples from rat or murine tissues were dilutedin sample/standard dilution buffer (PBS, 0.5% BSA, 15 ppm Proclin, 0.05%Tween 20, 0.25% CHAPS, 0.2% BgG, 5 mM EDTA, 0.35M NaCl, (pH 7.4)), addedto washed plates, and incubated for 1.5-2 hours. Plate-bound HtrA1 wasdetected during a 1-hour incubation with a biotinylated anti-muHtrA1rabbit polyclonal antibody (Genentech) diluted to 100 ng/mL for rattissues, and for mouse eye fluid and retina tissue, biotinylatedanti-huHtrA1 mouse monoclonal antibody (Genentech) was diluted to 200ng/mL, both in assay buffer (PBS, 0.5% BSA, 15 ppm Proclin, 0.05% Tween20), followed by a wash step and a 30-minute incubation withstreptavidin-HRP (GE Healthcare; Piscataway, N.J.), also diluted inassay buffer (1:20,000). After a final wash, tetramethyl benzidine (KPL,Gaithersburg, Md.) was added, color was developed for 10-15 minutes, andthe reaction was stopped with 1 M phosphoric acid. The plates were readat 450 nm with a 620 nm reference using a microplate reader. Theconcentrations of rat or murine HtrA1 were calculated from afour-parameter fit of the muHtrA1 standard curve.

As shown in FIG. 19A, expression of HtrA1 was low in rat ovary, brain,spleen, and total eye tissue as compared to the levels of expression ofHtrA1 in rat vitreous. As shown in FIG. 19B, the level of HtrA1 in mouseeye fluid increased in a model of constant light exposure as compared toa control. As shown in FIG. 19C, the level of HtrA1 in mouse retinaltissue increased in a model of constant light exposure as compared to acontrol.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. An isolated antibody that binds to HtrA1competitively with an antibody comprising a VH sequence of SEQ ID NO:8and a VL sequence of SEQ ID NO:7.
 2. The antibody of claim 1, whereincompetitive binding is determined using an ELISA assay.
 3. An isolatedantibody that binds to HtrA1, wherein the antibody (i) binds to anepitope that includes N224, K248, or both of HtrA1, and (ii) inhibitsHtrA1 with an IC₅₀ of ≤30 nM.
 4. The antibody of claim 3, wherein theantibody further comprises one or more of the following properties: (i)binds to HtrA1 with a ratio of 1 variable domain to one subunit of anHtrA1 trimer, or (ii) does not prevent trimer formation of HtrA1.
 5. Theantibody of claim 3, wherein the IC₅₀ is determined using a serineprotease assay with a substrate having SEQ ID NO:12.
 6. The antibody ofany one of claims 1-5, wherein the antibody does not cross-react withone or more of HtrA2, HtrA3 and HtrA4.
 7. The antibody of any one ofclaims 1-6, wherein the antibody has a dissociation constant of ≤500 nM.8. The antibody of claim 6, wherein the dissociation constant isdetermined by BIAcore using a Fab at 25° C.
 9. The antibody of any oneof claims 1-8, which is a monoclonal antibody.
 10. The antibody of anyone of claims 1-8, which is a human, humanized, or chimeric antibody.11. The antibody of any one of claims 1-8, which is an antibody fragmentthat binds HtrA1.
 12. The antibody of any one of claims 1-11, whereinthe antibody comprises (a) HVR-H3 comprising the amino acid sequenceGTFLTX_(p)WGHYFDY, wherein X_(p) is S or T (SEQ ID NO: 27); (b) HVR-L3comprising the amino acid sequence QQX_(g)X_(h)X_(i)X_(j)PX_(k)T,wherein X_(g) is S, V or D; X_(h) is Y, D or S; X_(i) is T, S, A, D orN; X_(j) is T, H, N, S, A, L or R; and X_(k) is P, T, A or S (SEQ ID NO:24); and (c) HVR-H2 comprising the amino acid sequenceWIDPYGGDTXoYADSVKG, wherein X_(o) is N or D (SEQ ID NO: 26).
 13. Theantibody of any one of claims 1-12, wherein the antibody comprises (a)HVR-H3 comprising the amino acid sequence of SEQ ID NO:6; (b) HVR-L3comprising the amino acid sequence of SEQ ID NO:3; and (c) HVR-H2comprising the amino acid sequence of SEQ ID NO:5.
 14. The antibody ofany one of claims 1-12, wherein the antibody comprises (a) HVR-H3comprising the amino acid sequence of SEQ ID NO:6; (b) HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:19; and (c) HVR-H2 comprising theamino acid sequence of SEQ ID NO:5.
 15. The antibody of any one ofclaims 1-12, wherein the antibody comprises (a) HVR-H3 comprising theamino acid sequence of SEQ ID NO:6: (b) HVR-L3 comprising the amino acidsequence of SEQ ID NO:22; and (c) HVR-H2 comprising the amino acidsequence of SEQ ID NO:5.
 16. The antibody of any one of claims 1-12,wherein the antibody comprises (a) HVR-H1 comprising the amino acidsequence GFX_(l)IX_(m)X_(n)YYIH, wherein X_(i) is N, S or T; X_(m) is S,D, Y or A; and X_(n) is G or D (SEQ ID NO: 25): (b) HVR-H2 comprisingthe amino acid sequence WIDPYGGDTX_(o)YADSVKG, wherein X_(o) is N or D(SEQ ID NO:26); and (c) HVR-H3 comprising the amino acid sequenceGTFLTX_(p)WGHYFDY, wherein X_(p) is S or T (SEQ ID NO: 27).
 17. Theantibody of any one of claims 1-12 or 16, further comprising (a) HVR-L1comprising the amino acid sequence RASQX_(a)X_(b)X_(c)X_(d)X_(e)X_(f)A,wherein X_(a) is D, S or V; X_(b) is V or I; X_(c) is S, N or G; X_(d)is T or N; X_(e) is A or Y; and X_(f) is V or L (SEQ ID NO: 23); (b)HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-L3comprising the amino acid sequence QQX_(g)X_(h)X_(i)X_(j)PX_(k)T,wherein X_(g) is S, V or D; X_(h) is Y, D or S; X_(i) is T, S, A, D orN: X_(j) is T, H, N, S, A, L or R; and X_(k) is P, T, A or S (SEQ ID NO:24).
 18. The antibody of any one of claims 1-12, 16 or 17, wherein theantibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQID NO:4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:5;and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6. 19.The antibody of claim 18, further comprising (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:1; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:3.
 20. The antibody of any one of claims 1-12, 16or 17, wherein the antibody comprises (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO:20; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO:5; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:6.
 21. The antibody of claim 20, furthercomprising (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO:18; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:19.
 22. Theantibody of claim 20, further comprising (a) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:21; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:22.
 23. The antibody of any one of claims 1-12, 16or 17, comprising (a) HVR-L1 comprising the amino acid sequence of SEQID NO:1; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:2;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:3. 24.The antibody of any one of claims 1-12, 16 or 17, comprising (a) HVR-L1comprising the amino acid sequence of SEQ ID NO:18; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:19.
 25. The antibody ofany one of claims 1-2, 16 or 17, comprising (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:21; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:2; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:22.
 26. The antibody of any one of claims 1-25,further comprising a heavy chain variable domain framework (FR2)sequence of SEQ ID NO:17.
 27. The antibody of any one of claims 1-26,comprising (a) a VH sequence having at least 95% sequence identity tothe amino acid sequence of SEQ ID NO:8; (b) a VL sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:7;or (c) a VH sequence as in (a) and a VL sequence as in (b).
 28. Theantibody of any one of claims 1-27, comprising (a) a VH sequencecomprising SEQ ID NO: 32; (b) a VL sequence comprising SEQ ID NO: 31; or(c) a VH sequence comprising SEQ ID NO: 32 and a VL sequence comprisingSEQ ID NO:
 31. 29. The antibody of claim 27 or 28, comprising a VHsequence of SEQ ID NO:8.
 30. The antibody of claim 27 or 28, comprisinga VL sequence of SEQ ID NO:7.
 31. The antibody of claim 27 or 28,comprising a VH sequence of SEQ ID NO:30.
 32. The antibody of claim 27or 28, comprising a VL sequence of SEQ ID NO:28.
 33. The antibody ofclaim 27 or 28, comprising a VL sequence of SEQ ID NO:29.
 34. Anantibody comprising a VH sequence of SEQ ID NO:8 and a VL sequence ofSEQ ID NO:7.
 35. An antibody comprising a VH sequence of SEQ ID NO:30and a VL sequence of SEQ ID NO:28.
 36. An antibody comprising a VHsequence of SEQ ID NO:30 and a VL sequence of SEQ ID NO:29.
 37. Anantibody that binds to HtrA1 comprising (a) a VH sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:8;(b) a VL sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO:7; or (c) a VH sequence as in (a) and a VLsequence as in (b).
 38. An antibody that binds to HtrA1 comprising (a) aVH sequence comprising SEQ ID NO: 32; (b) a VL sequence comprising SEQID NO: 31; or (c) a VH sequence comprising SEQ ID NO: 32 and a VLsequence comprising SEQ ID NO:
 31. 39. An antibody comprising (a) HVR-H1comprising the amino acid sequence GFX_(l)IX_(m)X_(n)YYIH, wherein X_(l)is N, S or T; X_(m) is S, D, Y or A; and X_(n) is G or D (SEQ ID NO:25); (b) HVR-H2 comprising the amino acid sequence WIDPYGGDTXoYADSVKG,wherein X_(o) is N or D (SEQ ID NO:26); (c) HVR-H3 comprising the aminoacid sequence GTFLTX_(p)WGHYFDY, wherein X_(p) is S or T (SEQ ID NO:27); (d) HVR-L1 comprising the amino acid sequenceRASQX_(a)X_(b)X_(c)X_(d)X_(e)X_(f)A, wherein X_(a) is D, S or V; X_(b)is V or I; X_(c) is S, N or G; X_(d) is T or N; X_(e) is A or Y; andX_(f) is V or L (SEQ ID NO: 23); (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2; and (f) HVR-L3 comprising the amino acidsequence QQX_(g)X_(h)X_(i)X_(j)PX_(k)T, wherein X_(g) is S, V or D;X_(h) is Y, D or S; X_(i) is T, S, A, D or N; X_(j) is T, H, N, S, A, Lor R; and X_(k) is P, T, A or S (SEQ ID NO: 24).
 40. The antibody ofclaim 39, wherein the antibody comprises (a) HVR-H1 comprising an aminoacid sequence selected from: SEQ ID NO:4, 20, and 47-51; (b) HVR-H2comprising an amino acid sequence selected from: SEQ ID NO:5 and 52; (c)HVR-H3 comprising an amino acid sequence selected from: SEQ ID NO:6 and53; (d) HVR-L1 comprising an amino acid sequence selected from: SEQ IDNO:1, 18, 21 and 33; (e) HVR-L2 comprising the amino acid sequence ofSEQ ID NO:2; and (f) HVR-L3 comprising an amino acid sequence selectedfrom: SEQ ID NO:3, 19, 22, and 34-46.
 41. The antibody of claim 40,wherein the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:4; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:5; (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO:6; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:1; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:3.
 42. The antibody ofclaim 40, wherein the antibody comprises (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO:20; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO:5; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO:6; (d) HVR-L1 comprising the amino acid sequence of SEQ IDNO:18; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:19.
 43. Theantibody of claim 40, wherein the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO:20; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:5; (c) HVR-H3 comprisingthe amino acid sequence of SEQ ID NO:6; (d) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:21; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO:2; and (f) HVR-L3 comprising the amino acidsequence of SEQ ID NO:22.
 44. The antibody of any one of claims 1-43,which is a full length IgG1 or IgG4 antibody.
 45. An isolated nucleicacid encoding the antibody of any one of claims 1-44.
 46. A host cellcomprising the nucleic acid of claim
 45. 47. A method of producing anantibody comprising culturing the host cell of claim 46 under conditionssuitable for expression of the nucleic acid encoding the anti-HtrA1antibody.
 48. The method of claim 47, further comprising recovering theanti-HtrA1 antibody from the host cell culture.
 49. An immunoconjugatecomprising the antibody of any one of claims 1-44 and a cytotoxic agent.50. A pharmaceutical formulation comprising the antibody of any one ofclaims 1-44 and a pharmaceutically acceptable carrier.
 51. The antibodyof any one of claims 1-44 for use as a medicament.
 52. The antibody ofany one of claims 1-44 for use in treating age-related maculardegeneration, geographic atrophy, diabetic retinopathy, retinopathy ofprematurity, or polypoidal choroidal vasculopathy.
 53. The antibody ofclaim 52, for use in treating dry age-related macular degeneration. 54.The antibody of any one of claims 1-44 for use in inhibitingdegeneration of retinal or photoreceptor cells.
 55. The antibody of anyone of claims 1-44 for use in inhibiting HtrA1 protease activity in aneye.
 56. Use of the antibody of any one of claims 1-44 in themanufacture of a medicament.
 57. The use of claim 56, wherein themedicament is for treatment of treating age-related maculardegeneration, geographic atrophy, diabetic retinopathy, retinopathy ofprematurity, or polypoidal choroidal vasculopathy.
 58. The use of claim57, wherein the age-related macular degeneration is dry age-relatedmacular degeneration.
 59. The use of claim 56, wherein the medicament isfor inhibiting degeneration of retinal or photoreceptor cells.
 60. Theuse of claim 56, wherein the medicament is for inhibiting HtrA1 proteaseactivity in an eye.
 61. A method of treating an individual havingage-related macular degeneration, geographic atrophy, diabeticretinopathy, retinopathy of prematurity, or polypoidal choroidalvasculopathy comprising administering to the individual an effectiveamount of the antibody of any one of claims 1-44.
 62. The method ofclaim 61, wherein the age-related macular degeneration is dryage-related macular degeneration.
 63. A method of inhibiting retinal orphotoreceptor cell degeneration in an individual comprisingadministering to the individual an effective amount of the antibody ofany one of claims 1-44 to inhibit retinal or photoreceptor celldegeneration.
 64. A method of inhibiting HtrA1 activity in an eye of anindividual comprising administering to the individual an effectiveamount of the antibody of any one of claims 1-44 to inhibit HtrA1activity in the eye.
 65. The antibody of any one of claims 1-12, whereinthe antibody comprises (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 87; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:88; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:89.
 66. The antibody of any one of claims 1-12 or 65, further comprising(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 85; (b)HVR-L2 comprising the amino acid sequence of SEQ ID NO:58; and (c)HVR-L3 comprising the amino acid sequence of SEQ ID NO:
 86. 67. Anantibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQID NO: 87; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:88; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:89;(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:85; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO:58; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:
 86. 68. Theantibody of claim 67, wherein the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:26; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:27; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:23; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:2; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:24.
 69. The antibody ofclaim 67 or 68, wherein the antibody comprises (a) HVR-H1 comprising anamino acid sequence selected from: SEQ ID NO:4, 20, 47-51 and 75-80; (b)HVR-H2 comprising an amino acid sequence selected from: SEQ ID NO:5, 52and 81-82; (c) HVR-H3 comprising an amino acid sequence selected from:SEQ ID NO:6, 53 and 83-84; (d) HVR-L1 comprising an amino acid sequenceselected from: SEQ ID NO:1, 18, 21, 33 and 54-57; (e) HVR-L2 comprisingan amino acid sequence selected from: SEQ ID NO:2 and 58; and (f) HVR-L3comprising an amino acid sequence selected from: SEQ ID NO:3, 19, 22,34-46 and 59-74.
 70. The antibody of any one of claims 65-69, which is afull length IgG1 or IgG4 antibody.
 71. An isolated nucleic acid encodingthe antibody of any one of claims 65-70.
 72. A host cell comprising thenucleic acid of claim
 71. 73. A method of producing an antibodycomprising culturing the host cell of claim 72 under conditions suitablefor expression of the nucleic acid encoding the anti-HtrA1 antibody. 74.The method of claim 73, further comprising recovering the anti-HtrA1antibody from the host cell culture.
 75. An immunoconjugate comprisingthe antibody of any one of claims 65-70 and a cytotoxic agent.
 76. Apharmaceutical formulation comprising the antibody of any one of claims65-70 and a pharmaceutically acceptable carrier.
 77. The antibody of anyone of claims 65-70 for use as a medicament.
 78. The antibody of any oneof claims 65-70 for use in treating age-related macular degeneration,geographic atrophy, diabetic retinopathy, retinopathy of prematurity, orpolypoidal choroidal vasculopathy.
 79. The antibody of claim 78, for usein treating dry age-related macular degeneration.
 80. The antibody ofany one of claims 65-70 for use in inhibiting degeneration of retinal orphotoreceptor cells.
 81. The antibody of any one of claims 65-70 for usein inhibiting HtrA1 protease activity in an eye.
 82. Use of the antibodyof any one of claims 65-70 in the manufacture of a medicament.
 83. Theuse of claim 82, wherein the medicament is for treatment of treatingage-related macular degeneration, geographic atrophy, diabeticretinopathy, retinopathy of prematurity, or polypoidal choroidalvasculopathy.
 84. The use of claim 83, wherein the age-related maculardegeneration is dry age-related macular degeneration.
 85. The use ofclaim 82, wherein the medicament is for inhibiting degeneration ofretinal or photoreceptor cells.
 86. The use of claim 82, wherein themedicament is for inhibiting HtrA1 protease activity in an eye.
 87. Amethod of treating an individual having age-related maculardegeneration, geographic atrophy, diabetic retinopathy, retinopathy ofprematurity, or polypoidal choroidal vasculopathy comprisingadministering to the individual an effective amount of the antibody ofany one of claims 65-70.
 88. The method of claim 87, wherein theage-related macular degeneration is dry age-related maculardegeneration.
 89. A method of inhibiting retinal or photoreceptor celldegeneration in an individual comprising administering to the individualan effective amount of the antibody of any one of claims 65-70 toinhibit retinal or photoreceptor cell degeneration.
 90. A method ofinhibiting HtrA1 activity in an eye of an individual comprisingadministering to the individual an effective amount of the antibody ofany one of claims 65-70 to inhibit HtrA1 activity in the eye.